doi:10.1152/ajprenal.00329.

2010
300:F1076-F1088, 2011. First published 9 February 2011; Am J Physiol Renal Physiol
Zhuo
Xiao C. Li, Julia L. Cook, Isabelle Rubera, Michel Tauc, Fan Zhang and Jia L.
increases blood pressure in rats and mice
fusion of angiotensin II selectively in proximal tubules
Intrarenal transfer of an intracellular fluorescent
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Intrarenal transfer of an intracellular uorescent fusion of angiotensin II
selectively in proximal tubules increases blood pressure in rats and mice
Xiao C. Li,
1
Julia L. Cook,
2
Isabelle Rubera,
3
Michel Tauc,
3
Fan Zhang,
4
and Jia L. Zhuo
1,4
1
Laboratory of Receptor and Signal Transduction, Department of Pharmacology and Toxicology, University of Mississippi
Medical Center, Jackson, Mississippi;
2
Ochsner Clinic Foundation, New Orleans, Louisiana;
3
UMR-CNRS 6548, University
of Nice-Sophia Antipolis, Parc Valrose, Nice, France; and
4
Division of Hypertension and Vascular Research, Department
of Internal Medicine, Henry Ford Hospital, Detroit, Michigan
Submitted 8 June 2010; accepted in nal form 3 February 2011
Li XC, Cook JL, Rubera I, Tauc M, Zhang F, Zhuo JL.
Intrarenal transfer of an intracellular uorescent fusion of angiotensin
II selectively in proximal tubules increases blood pressure in rats and
mice. Am J Physiol Renal Physiol 300: F1076F1088, 2011. First
published February 9, 2011; doi:10.1152/ajprenal.00329.2010.The
present study tested the hypothesis that intrarenal adenoviral transfer
of an intracellular cyan uorescent fusion of angiotensin II (ECFP/
ANG II) selectively in proximal tubules of the kidney increases blood
pressure by activating AT
1
(AT
1a
) receptors. Intrarenal transfer of
ECFP/ANG II was induced in the supercial cortex of rat and mouse
kidneys, and the sodium and glucose cotransporter 2 (sglt2) promoter
was used to drive ECFP/ANG II expression selectively in proximal
tubules. Intrarenal transfer of ECFP/ANG II induced a time-depen-
dent, proximal tubule-selective expression of ECFP/ANG II in the
cortex, which peaked at 2 wk and was sustained for 4 wk. ECFP/ANG
II expression was low in the glomeruli and the entire medulla and was
absent in the contralateral kidney or extrarenal tissues. At its peak of
expression in proximal tubules at day 14, ANG II was increased by
twofold in the kidney (P 0.01) and more than threefold in proximal
tubules (P 0.01), but remained unchanged in plasma or urine.
Systolic blood pressure was increased in ECFP/ANG II-transferred
rats by 28 6 mmHg (P 0.01), whereas fractional sodium
excretion was decreased by 20% (P 0.01) and fractional lithium
excretion was reduced by 24% (P 0.01). These effects were blocked
by losartan and prevented in AT
1a
knockout mice. Transfer of a
scrambled ECFP/ANG IIc had no effects on blood pressure, kidney,
and proximal tubule ANG II, or sodium excretion. These results
provide evidence that proximal tubule-selective transfer of an intra-
cellular ANG II fusion protein increases blood pressure by activating
AT
1a
receptors and increasing sodium reabsorption in proximal tu-
bules.
adenoviral gene delivery; G protein-coupled receptors; intracrine
peptides; losartan; renin-angiotensin-aldosterone system; urinary so-
dium excretion
THE RENIN-ANGIOTENSIN SYSTEM (RAS) is long recognized to exist
and function as dual extracellular (endocrine and/or paracrine)
and intracellular (or intracrine) vasoactive hormonal systems.
The extracellular system includes circulating and local tissue
ANG II, which plays the classic roles of ANG II through
activation of cell surface G protein-coupled ANG II receptors
(GPCRs) (6, 21, 30, 35). The intracellular system includes
intracellularly formed ANG II (15, 810, 12, 19, 40) and
extracellular ANG II internalized through type 1 (AT
1
) recep-
tor-mediated endocytosis (14, 15, 18, 19, 25, 27, 39). The roles
of circulating and paracrine ANG II and G protein-coupled
signaling transduction mechanisms via activation of cell sur-
face receptors have been extensively studied (6, 21, 30, 35). By
contrast, whether intracellular ANG II may induce any physi-
ological effects remains largely unknown. The slow progress in
our understanding of physiological roles of intracellular ANG
II has been stymied in part due to the lack of suitable animal
models that express an ANG II peptide in a particular tissue or
cell, which is produced intracellularly but not released or
secreted into the extracellular uid compartment or the circu-
lation, and the technical challenges in distinguishing between
the effects induced by intracellular vs. extracellular ANG II
through activation of its intracellular vs. cell surface receptors.
Transgenic mice have been generated to express an ANG
II-producing fusion protein in cardiomyocytes using the -my-
osin heavy chain promoter (32, 34). This cardiac-specic ANG
II construct contains a signal peptide sequence from human
prorenin and a furin cleavage site. ANG II fusion protein can
be cleaved by furin, released into the secretory pathway, and
secreted into the cardiac interstitium (32, 34). Thus this ANG
II fusion protein activates cell surface rather than intracellular
or nuclear receptors to induce paracrine effects similar to
extracellular ANG II. Alternatively, an adenoviral vector en-
coding an intracellular ANG II peptide has been used to study
the hypertrophic effect of cardiac-specic ANG II (1). Intra-
cardiac transduction of this peptide in mice resulted in cardiac
hypertrophy without affecting blood pressure and circulating
ANG II levels. However, the cardiac hypertrophic effect in-
duced by this intracellular ANG II is not blocked by the AT
1
receptor blocker losartan, suggesting an AT
1
receptor-indepen-
dent effect of this novel intracellular ANG II peptide. Most
recently, transgenic mice expressing an intracellular uores-
cent fusion of ANG II have been generated using the mouse
metallothionein promoter (28). Global expression of this trans-
gene, enhanced cyan uorescent protein (ECFP)/ANG II, in all
tissues, including brain, heart, kidney, liver, lung, and testes,
shows a blood pressure-elevating effect and renal microangi-
opathy (28).
In the present study, we developed an adenoviral construct
encoding a uorescent fusion of ANG II (ECFP/ANG II),
which is linked by a small spacer arm, upstream and in-frame,
to ECFP (3, 4, 28). The fusion protein is designed in a manner
that ensures ECFP/ANG II to be synthesized on free ribo-
somes, but not destined to the secretory pathway for secretion
out of the cells (4, 28). The sodium and glucose cotransporter
2 (sglt2) promoter was used to drive the expression of ECFP/
ANG II selectively in proximal tubules of rat and mouse
kidneys (29). We report here for the rst time that intrarenal
adenoviral transfer of this intracellular ANG II fusion protein
Address for reprint requests and other correspondence: J. L. Zhuo, Dept. of
Pharmacology and Toxicology, Univ. of Mississippi Medical Center, 2500
North State St., Jackson, MS 39216-4505 (e-mail: jzhuo@umc.edu).
Am J Physiol Renal Physiol 300: F1076F1088, 2011.
First published February 9, 2011; doi:10.1152/ajprenal.00329.2010. Innovative Methodology
1931-857X/11 Copyright 2011 the American Physiological Society http://www.ajprenal.org F1076

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selectively in proximal tubules of rat and mouse kidneys can
increase blood pressure and decreased fractional sodium and
lithium excretion through AT
1a
receptor-dependent mecha-
nisms.
METHODS
Construction of the ECFP/ANG II transgene. The expression plas-
mid encoding a cyan uorescent fusion of ANG II, ECFP/ANG II, and
a scrambled version of the plasmid were generated by Dr. Julie Cook
of the Ochsner Clinic Foundation. Construction of ECFP/ANG II or
its scrambled version, ECFP/ANG IIc, was previously described (4).
Briey, ANG II or ANG IIc was ligated downstream of ECFP in the
cyan uorescent fusion vector pECFP-C1 (Clontech, Palo Alto, CA) to
generate a cyan uorescent fusion protein, ECFP/ANG II or ECFP/ANG
IIc. The upstream primer 5=-AGCTTCAGACCGCGTATACATCCAC-
CCCTTTTAGG-3= and downstream primer 5=-GATCCCTA-
AAAGGGGTGGATGTATACGCGGTCTGA-3= were annealed, di-
gested with HindIII and BamHI, and cloned into HindIII/BamHI-digested
pECFP-C1. For ECFP/ANG IIc, the upstream primer was 5=-
AGCTTCATACGACCACCGCGTATTTCCCATCTAGG-3=, and the
downstream primer was 5=-GATCCCTAGATGGGAAATACGCG-
GTGGTCGTATGA-3= (4). The cloned plasmids were further sequenced
for conrmation.
Construction of the ECFP/ANG II transgene with the proximal
tubule-specic promoter sglt2. The sglt2 promoter was provided by
Drs. Isabelle Rubera and Michel Tauc of the University of Nice-
Sophia Antipolis (Nice, France) and was used to drive the expression
of ECFP/ANG II or ECFP/ANG IIc selectively in proximal tubules of
the kidney (29). The promoter contains 2,637 bp of the murine sglt2
5=-anking region (nucleotides 55-2691 of GenBank accession no.
AJ292928, exon 1 within the initiation codon ATG, intron 1, and the
rst part 923 bp of exon 2) (24). To enable proximal tubule-specic
expression of ECFP/ANG II or ECFP/ANG IIc using the sglt2
promoter, the NotI fragment (2.6 kb) of pGEM-sglt25pr-mut was
rst subcloned into the NotI-digested DUAL-Basic vector. The NheI/
AII/Klenow fragment (1.1 kb) of pECFP/ANG II was then subcloned
into XhoI/Klenow-digested DUAL-sglt25pr-mut (Vector BioLab,
Philadelphia, PA). The entire expression cassette was then transferred
to the adenovirus genome vector and conrmed through restriction
mapping with a titer of 4.6 10
11
pfu/ml. The specic adenoviral
vector used in this study was Ad-sglt2-ECFP/ANG II, and the control
vector was Ad-sglt2-ECFP/ANG IIc (Fig. 1). The specicity of the
sglt2 promoter to drive specic expression of Cre recombinase in the
kidney proximal tubules has been conrmed in a transgenic Cre/Lox
mouse model (29).
Expression of ECFP/AII in cultured mouse proximal tubule cells.
Semiconuent mouse proximal tubule cells (a gift from Dr. Ulrich
Hopfer, Case Western Reserve University) in six-well plates or on
glass coverslips were transfected with the specic transgene Ad-sglt2-
ECFP/ANG II or scrambled transgene Ad-sglt2-ECFP/ANG IIc (4
g/well) for 48 h using the transfection protocol as we described
previously (14, 17, 40). The medium was collected for measurement
of ECFP as a marker of expression using a uorescent plate reader,
whereas the cells were visualized using a Nikon-Eclipse TE2000-U
inverted uorescence microscope and a dual 4=,6-diamidino-2-phe-
nylindole (DAPI)-CFP lter set (excitation: 440 nm; emission: 480
nm). In further experiments, ANG II peptides were extracted from the
medium and proximal tubule cells for measurements of ANG II levels
as we described previously (14, 17, 19, 40).
Intrarenal adenoviral transfer of ECFP/ANG II or ECFP/ANG IIc.
Seven groups (n 616 each) of adult male Sprague-Dawley rats (74
in total) and two groups (n 8 each) of wild-type (C57BL/6J; 16 in
total) and AT1a
receptor-decient mice (Agtr1a/ or AT
1a
-KO; 16
in total) were used in the current study. All animals were maintained
on a normal ration of rodent chow and had free access to tap water.
Basal systolic blood pressure (SBP), 24-h drinking, urine, and urinary
sodium excretion were rst determined before intrarenal transfer of
ECFP/ANG II or ECFP/ANG IIc was performed. To induce intrarenal
adenoviral ECFP/ANG II transfer, rats and wild-type or AT
1a
-KO
mice were anesthetized and their left renal artery was temporarily
clamped with a ne vessel clip, which briey interrupted blood ow
to the left kidney for 5 min. Ad-sglt2-ECFP/ANG II or Ad-sglt2-
ECFP/ANG IIc was diluted 1:5 in phosphate-buffered saline and
directly injected into the supercial cortex evenly with six locations
(20 l each) (22, 23). By contrast, the control groups of rats or mice
received sham injections of saline instead. Blood ow to the left
kidney was reestablished 5 min after injection of Ad-sglt2-ECFP/
ANG II or Ad-sglt2-ECFP/ANG IIc. The duration of renal blood ow
disruption for a brief 5 min was chosen and based on a series of
preliminary time-dependent studies (515 min). No long-term histo-
logical or pathological changes were identied while effective intrare-
nal adenoviral gene transfer with renal blood ow interruption for 5
min was ensured. Similar ndings were reported previously in rats
(22, 23). Animals were then maintained for 1 wk or up to 4 wk
dependent on a particular experimental protocol. To determine the
role of AT
1
receptors in mediating the effects of ECFP/ANG II
transfer in rats, a group of rats was transferred with ECFP/ANG II in
proximal tubules and concurrently treated with the AT
1
receptor
blocker losartan (20 mgkg
1
day
1
po) for 2 wk. All experiments
using animals were approved by the Institutional Animal Care and
Use and Recombinant DNA and Biosafety Committees of the Henry
Ford Health System (Detroit, MI) and the University of Mississippi
Medical Center (Jackson, MS), respectively.
Measurement of blood pressure and 24-h drinking and urinary
excretion of water and electrolytes. Basal and weekly systolic blood
pressure in rats and mice was measured for 14 wk using the tail-cuff
method as we described previously (15, 16, 18, 38). Basal and weekly
24-h drinking, urine, and urinary sodium and potassium excretion
were measured using a metabolic cage and a NOVA 13 electrolyte
analyzer (Nova Biomedicals) in all animals maintained on the same
rations of chow and water as we described previously for 14 wk (15,
16, 18, 38).
Measurement of plasma and urine creatinine and lithium concentrations.
Plasma and urine creatinine concentrations were determined using a
creatinine parameter colorimetric assay kit (R&D Systems, Minneap-
olis, MN) (11, 20). To measure lithium clearance as an indirect index
of proximal tubule sodium reabsorption in the rat kidney, animals
were fed a chow containing 15 mmol/kg dry wt food throughout the
experiment as described elsewhere (36, 37). Lithium concentrations
were measured using a NOVA 13 electrolyte analyzer (Nova Bio-
medicals). Creatinine and lithium clearance were calculated using the
standard renal clearance method (11, 20, 36, 37).
Measurements of plasma, kidney, proximal tubule, and urine ANG
II levels. At the end of experiment, rats were decapitated without
anesthesia, and trunk blood samples collected to measure plasma
ECFP and ANG II levels as described (15, 18, 24, 38). The left kidney
was sliced through the middle into two portions, with one-half
Fig. 1. Schematic construction map of recombinant human adenoviral vectors
carrying the cyan uorescent fusion of ANG II (ECFP/ANG II) or its
scrambled control ANG IIc (ECFP/ANG IIc) and the sodium and glucose
cotransporter 2 (sglt2) gene promoter.
Innovative Methodology
F1077 INTRACELLULAR ANGIOTENSIN II ON BLOOD PRESSURE
AJP-Renal Physiol VOL 300 MAY 2011 www.ajprenal.org

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immediately processed for measurement of the whole kidney ANG II
levels as described (15, 18, 24, 38). The other half of the left kidney
was used to isolate fresh proximal tubules from the supercial cortex
by collagenase digestion, sequential ltrations, and separation on a
50% Percoll gradient (33). In separate experiments, ve rat supercial
cortex samples were dissected on ice into three portions to determine
the rate of ANG II degradation due to collagenase digestion, ltration,
and separation of proximal tubules. The rst portion was immediately
processed for measuring renal cortical ANG II levels without being
processed through collagenase digestion and sequential separation
procedures. ANG II levels measured as such were taken as an indirect
index of proximal tubule ANG II without degradation, because prox-
imal tubules account for 90% of tissues in the supercial cortex. The
second portion was digested with collagenase and left at 37C in a
buffer containing an inhibitor cocktail for 45 min, the time required
for the isolation of proximal tubules. The differences in ANG II levels
between the rst and second portions of cortical samples represent the
ANG II levels degraded during the proximal tubule isolation pro-
cesses. The third portion of samples was used for collagenase diges-
tion at 37C and sequential separation of proximal tubules in a buffer
containing an inhibitor cocktail. ANG II levels measured as such
represent intracellular ANG II levels in proximal tubules following
collagenase digestion and degradation (Table 1). Plasma, kidney, and
proximal tubule ANG II were measured using a sensitive ELISA kit
(Bachem) (14, 15, 17, 18). Proximal tubule ANG II levels were
corrected by the 42% of ANG II degradation rate during collagenase
digestion and sequential separation of proximal tubules. In a recent
study, van Esch et al. (31) showed that close to 70% of ANG II in the
kidney was metabolized if the samples were simply left at room
temperature. ANG II was also extracted from urine samples using a
phenyl-bonded solid-phase peptide extraction column (Elut-C18, Var-
ian), vacuum-dried overnight, and reconstituted in an ANG II assay
buffer. Urinary ANG II was measured as described for plasma ANG
II and corrected by 24-h urine excretion.
Fluorescent microscopic imaging of ECFP/ANG II or ECFP/ANG
IIc expression in the kidney and extrarenal tissues. To conrm the
proximal tubule-specic expression and to exclude the ectopic expres-
sion of ECFP/ANG II or ECFP/ANG IIc in the kidney and/or
extrarenal tissues, fresh and frozen sections (6-m thick) were cut on
a cryostat and thaw-mounted on glass slides from adrenal glands,
brain, heart, kidney, liver, lung, and skeletal smooth muscles of
separate groups of rats with or without intrarenal ECFP/ANG II or
ECFP/ANG IIc transfer. Sections were then briey counterstained
with the cell nuclear marker DAPI (300 nM) for 5 min, washed with
phosphate-buffered saline, and mounted on a microscopic stage.
Expression of ECFP/ANG II or ECFP/ANG IIc in these tissues was
visualized using a Nikon-Eclipse TE2000-U inverted uorescence
microscope and a dual DAPI-CFP band-pass excitation lter set
(excitation: 440 nm; emission: 495/50 nm). Nuclear DAPI-stained
images were converted into red for better differentiation between
ECFP (blue-green) and DAPI (blue). In all uorescence imaging
analyses, the background autouorescence level was determined in
the renal medulla of the same kidney with ECFP/ANG II or ECFP/
ANG IIc transfer in the supercial cortex or in the contralateral kidney
that was not transferred with ECFP/ANG II or ECFP/ANG IIc.
Fluorescent immunohistochemistry of CFP and ANG II in the
kidney. To provide additional evidence for the increased expression of
ECFP and ANG II specically in proximal tubules of the kidney,
uorescent immunohistochemistry was performed using a mouse
monoclonal anti-cyan uorescence protein (CFP) antibody (1:250,
Abm, Richmond, ON) or a rabbit anti-human ANG II antibody (1:250,
USBiological, Swampscott, MA), respectively. Secondary antibodies
used were FITC-conjugated donkey anti-mouse or anti-rabbit anti-
body (1:2,000, Santa Cruz Biotechnology, Santa Cruz, CA), respec-
tively. Fluorescent immunostaining for CFP or ANG II was visualized
using a Nikon-Eclipse TE2000-U inverted uorescence microscope
and a dual DAPI-FITC band-pass excitation lter set (excitation: 488
nm; emission: 510/50 nm). For better image visualization or differ-
entiation, nuclear DAPI staining was converted to red, ANG II
immunostaining to green, and CFP immunostaining to blue-green
images, respectively.
Statistical analysis. All results are presented as means SE.
One-way ANOVA was rst to compare the differences in the same
parameters between groups of rats or mice. If the P value was 0.05,
a post hoc Newman-Keuls multiple comparison test was performed
to compare two different group means. The signicance was set at
P 0.05.
RESULTS
Expression of ECFP/ANG II in cultured proximal tubule
cells. Figure 2 shows that transfection of mouse proximal
tubule cells with the adenoviral construct Ad-sglt2-ECFP/ANG
II (4 g/well) for 48 h induced intensive expression of ECFP/
ANG II throughout the cytoplasm and perinuclear regions of
the cells. Expression of ECFP/ANG II increased intracellular
ANG II levels by more than twofold (control: 191.9 17.6 vs.
ECFP/ANG II: 484.0 31.8 pg/mg protein, **P 0.01).
Expression of ECFP/ANG IIc did not alter proximal tubule
ANG II levels (246.4 37.9 pg/mg protein, not signicant vs.
control). By contrast, ANG II levels in the medium remained
very low (26.1 3.8 pg/mg protein). There were no signicant
differences in the medium ECFP levels between control cells
and cells transfected with either ECFP/ANG II or ECFP/ANG
IIc (not shown).
Proximal tubule-specic expression of ECFP/ANG II in the
kidney. Intrarenal adenoviral transfer of ECFP/ANG II using
the sglt2 promoter led to a time-dependent expression of the
transgene (cyan uorescence as blue-green) selectively in
proximal tubules throughout the supercial cortex (Fig. 3,
AI). Close visualization of serial longitudinal sections of the
kidneys that were transferred with ECFP/ANG II (n 6 for
each time point) showed that the expression of ECFP/ANG II
reached 78 6% of proximal tubules in the cortex 7 days after
intrarenal transfer (Fig. 3A). The proximal tubule-specic ex-
pression of ECFP/ANG II was increased further to 92 3% by
2 wk (Fig. 3D) and persisted for 4 wk after transfer (Fig. 3G).
There were very low levels of ECFP/ANG II expression in the
glomeruli (Fig. 3, A, D, and G) or in the outer renal medulla in
the same kidney that was transferred with ECFP/ANG II in the
supercial cortex (Fig. 3, JR). Figure 4 compares ECFP/ANG
II expression between the left kidney with the ECFP/ANG II
transfer and the contralateral right kidney without ECFP/ANG
II transfer. While ECFP/ANG II expression was observed
selectively in all proximal tubules of the left kidney (Fig. 4A),
Table 1. Effects of collagenase digestion and proximal
tubule isolation procedures on ANG II degradation or
metabolism in the rat kidney
Supercial Cortex
(0C)
Supercial Cortex
(37C)
Isolated Proximal
Tubule (37C)
ANG II, pg/mg protein 129.2 11.3 75.6 6.8* 78.0 14.5*
Values are means SE. Supercial cortical samples was taken as an
indirect index of proximal tubule samples, because proximal tubules account
for 90% of tissues in the supercial cortex. Note that ANG II level was
decreased by 42% in supercial cortical samples subjected to collagenase
digestion and proximal tubule isolation buffer at 37C. *P 0.01 vs.
supercial cortical samples immediately homogenized in an ANG II extraction
buffer on ice and extracted for ANG II assays (0C) (15, 18, 38).
Innovative Methodology
F1078 INTRACELLULAR ANGIOTENSIN II ON BLOOD PRESSURE
AJP-Renal Physiol VOL 300 MAY 2011 www.ajprenal.org

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no signicant ECFP/AII expression was seen in proximal
tubules of the contralateral right kidney (Fig. 4C). Similarly,
very low levels of ECFP/ANG II expression were expressed in
the inner medulla of left (Fig. 4B) and right kidneys (Fig. 4D).
Furthermore, ECFP/AII expression was seen throughout the
tubule wall in freshly isolated proximal tubules (Fig. 5, A and
C), whereas no signicant ECFP/ANG II expression was
observed in freshly isolated glomeruli (Fig. 5, D and F).
To further conrm the increased expression of ECFP and
ANG II in proximal tubules of the rat kidney with ECFP/ANG
II transfer, uorescent immunohistochemistry was performed
using a specic anti-CFP or anti-ANG II antibody (Fig. 6).
Increased anti-CFP (blue-green, Fig. 6, A and C) and anti-ANG
II (green, Fig. 6, D and F) immunouorescence staining was
observed in all proximal tubules of the kidneys with ECFP/
ANG II transfer. In the control rat kidney without ECFP/ANG
II transfer, little anti-CFP immunouorescence staining was
observed in the glomeruli and proximal tubules (Fig. 6G).
However, anti-ANG II immunostaining was seen in proximal
tubules of the control kidney, but the level was much lower
than that found in the ECFP/ANG II-transferred kidney (Fig. 6,
D and F). The relative time-related expression of ECFP/ANG
II in the glomeruli, proximal tubules, and the medulla of the
kidney is summarized in Table 2.
For a direct comparison, proximal tubule-specic expres-
sion of the scrambled version of the fusion protein ECFP/
ANG IIc in the rat kidney is shown in Fig. 7. The pattern of
ECFP/ANG IIc expression or its distribution in proximal
tubules of the kidney was similar to that of ECFP/ANG II
(Figs. 5 and 6).
Ectopic expression of ECFP/ANG II in extrarenal tissues.
To determine whether intrarenal transfer of ECFP/ANG II
escapes into the circulation and induces ectopic expression in
other extrarenal tissues, the heart, liver, spleen, brain, lung, and
adrenals were examined for ECFP/ANG II expression. Figure 8
shows that intrarenal adenoviral transfer of ECFP/ANG II
using the sglt2 promoter did not lead to signicant ectopic
expression of the transgene in all extrarenal tissues we exam-
ined.
Effects of proximal tubule-specic transfer of ECFP/ANG II
on plasma, kidney, proximal tubule, and urine ANG II levels.
As shown in Fig. 9A, plasma ANG II levels were not different
between control and ECFP/ANG II-transferred rats (control:
228.9 30.9 vs. ECFP/ANG II: 247.6 36 fmol/ml, not
signicant). By contrast, ANG II levels in the kidney were
increased signicantly by more than twofold in rats 2 wk after
intrarenal ECFP/ANG II transfer (control: 415.3 49.3 vs.
ECFP/ANG II: 857.6 80.2 pg/g kidney wt, P 0.01) (Fig.
9B). Proximal tubule ANG II levels were increased by more
than threefold from 73.8 10.2 pg/mg protein in control rats
to 252.7 13.2 pg/mg protein in ECFP/ANG II-transferred
rats (P 0.01) (Fig. 9C). Concurrent treatment with losartan in
ECFP/AII-transferred rats signicantly reduced kidney ANG II
(643.2 48.0 pg/g kidney wt, P 0.05) and proximal tubule
ANG II (115.9 12.3 pg/mg protein, P 0.01) to levels lower
than those of ECFP/ANG II-transferred but higher than those
of control rats. By contrast, plasma ANG II levels in ECFP/
ANG II-transferred rats treated with losartan were increased by
about threefold over control rats in part due to inhibition of
AT
1
receptor-mediated uptake of circulating ANG II in tissues
(ECFP/ANG IIlosartan: 705.1 pg/ml, P 0.01). There
were no signicant differences in 24-h urinary ANG II excre-
tion between the ECFP/ANG II-transferred and nontransferred
rats (control: 466.8 46.3 vs. ECFP/ANG II: 492.1 36.5
pg/24 h, not signicant) (Fig. 9D).
Intrarenal adenoviral transfer of the scrambled version of the
fusion protein ECFP/ANG IIc had no signicant effects on
plasma, kidney, proximal tubule, and urinary ANG II levels
compared with control rats (Fig. 9).
Fig. 2. Expression of an intracellular cyan u-
orescent fusion of ANG II, ECFP/ANG II, in
cultured mouse proximal tubule cells 48 h after
transfection. A: expression of ECFP/ANG II in
the cytoplasm and perinuclear region, shown as
blue-green. B: 4=,6-diamidino-2-phenylindole
(DAPI)-stained nuclei, which was converted to
red to facilitate visualization. C: merged image
of A and B. D: ANG II levels in non-ECFP/
ANG II-transfected (control), ECFP/ANG II-
transfected, and scrambled ECFP/ANG IIc-
transfected cells, and in the culture medium.
Bar 20 m.
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Effect of proximal tubule-specic transfer of ECFP/ANG II
selectively in proximal tubules on systolic blood pressure in
rats. Baseline systolic blood pressure was similar in all groups
of animals before ECFP/ANG II transfer was performed,
which remained unchanged throughout the study in sham
control rats (Fig. 10A). In rats with intrarenal transfer of
ECFP/ANG II selectively in proximal tubules, systolic blood
pressure increased from a baseline of 118 4 to 137 4
mmHg at day 7 (P 0.01) and further to 149 3 mmHg at
day 14 after ECFP/ANG II transfer (P 0.01). Concurrent
treatment with losartan in rats treated with ECFP/ANG II
transfer prevented the increases in blood pressure induced by
ECFP/ANG II (Fig. 10A). Intrarenal adenoviral transfer of the
control ECFP/ANG IIc had no effect on blood pressure in rats
(Fig. 10A). In further experiments in separate groups of time-
control (n 5) and ECFP/ANG II-transferred rats (n 7),
systolic blood pressure was monitored at their baselines and
then weekly for 4 wk. Systolic blood pressure remained ele-
vated 4 wk after ECFP/ANG II transfer in the kidney (see
Table 4).
Fig. 3. Time-dependent expression of ECFP/
ANG II in proximal tubules of the rat renal
cortex (AI), but not in the outer medulla
(JR), 7, 14, or 28 days after intrarenal ad-
enoviral transfer of ECFP/ANG II in the
supercial cortex of the kidney. A, D, G, J,
M, and P: ECFP/ANG II expression as blue-
green. B, E, H, K, N, and Q: nuclear DAPI
staining, which was converted to red to fa-
cilitate visualization. C, F, I, L, O, and
R: merged or overlaid images of ECFP/ANG
II and DAPI staining. G, glomerulus; PT,
proximal tubule. Bars 100 m.
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Effect of proximal tubule-specic transfer of ECFP/ANG II
on blood pressure in AT
1a
-KO mice. In wild-type C57BL/6J
mice, intrarenal adenoviral ECFP/ANG II transfer signicantly
increased systolic blood pressure at day 14 (control: 122 2
vs. ECFP/ANG II: 154 3 mmHg, P 0.01) (Fig. 10B).
However, intrarenal adenoviral ECFP/ANG II transfer had no
signicant effect on blood pressure in AT
1a
-KO mice (control:
98 3 vs. ECFP/ANG II: 102 8 mmHg, not signicant)
(Fig. 10B).
Effects of proximal tubule-specic transfer of ECFP/ANG II
or ECFP/ANG IIc on body, heart, and kidney weights and 24-h
urine excretion and fractional sodium and lithium excretion.
Proximal tubule expression of ECFP/ANG II or ECFP/ANG
IIc did not signicantly alter the left kidney weight-to-body
weight or the heart weight-to-body weight ratio (Table 2).
ECFP/ANG II, but not ECFP/ANG IIc, expression in proximal
tubules decreased 24-h urine and urinary sodium and potas-
sium excretion 2 wk after ECFP/ANG II transfer, and the
responses were reversed by concurrent treatment with losartan
(Table 3). Fractional sodium (control: 0.18 0.01 vs. ECFP/
ANG II: 0.14 0.010%, P 0.01) and lithium excretion
(control: 0.33 0.02 vs. ECFP/ANG II: 0.25 0.03%., P
0.01) was signicantly decreased by intrarenal ECFP/ANG II
transfer. Concurrent treatment with losartan reversed the frac-
tional sodium excretion (0.20 0.01%, P 0.01 vs. ECFP/
ANG II) as well as fractional lithium excretion (0.40 0.02%,
P 0.01 vs. ECFP/ANG II) to their control levels 2 wk after
proximal tubule-specic transfer (Fig. 11). However, 24-h
Fig. 5. Expression of ECFP/ANG II selectively in freshly isolated proximal tubules of the rat kidney 2 wk after intrarenal ECFP/ANG II transfer. Bars 10
m for the proximal tubule or 30 m for the glomerulus.
Fig. 4. Peak proximal tubule-selective expres-
sion of ECFP/ANG II in the rat kidney 2 wk
after intrarenal adenoviral transfer. A: specic
ECFP/ANG II expression in proximal tubules
of the left kidney, which received ECFP/ANG
II transfer in the supercial cortex. B: lack of
ECFP/ANG II expression in the left inner me-
dulla. C: lack of ECFP/ANG II expression in
the right contralateral renal cortex. D: lack of
ECFP/ANG II expression in the right contralat-
eral inner medulla. Images shown are merged
from cyan (ECFP/ANG II) and DAPI uores-
cence (nuclei). G, glomerulus. PT, proximal
tubules. Bar 100 m.
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urinary excretion of water and sodium returned to the levels
signicantly higher than those of time control rats 28 days after
ECFP/ANG II transfer in the presence of continuously elevated
blood pressure (Table 4).
DISCUSSION
Although intracellularly administered ANG II can induce
biological effects in cultured cells or in freshly isolated nuclei
(2, 4, 810, 19, 25, 26), whether intracellular ANG II plays a
physiological role in the regulation of blood pressure and renal
function is unknown. The goal of the current study was to test
the hypothesis that intrarenal adenovirus-mediated transfer of
an intracellular cyan uorescent fusion of ANG II (ECFP/ANG
II) selectively in proximal tubules of the rat and mouse kidneys
increases arterial blood pressure by activating AT
1
(AT
1a
)
receptors in the kidney. Our current results demonstrate that
proximal tubule cell-specic transfer of this intracellular ANG
II fusion protein indeed increases systolic blood pressure in rats
Fig. 6. Immunouorescence staining for increased expression of ECFP and ANG II in proximal tubules of the rat kidney transferred with ECFP/ANG II for 2
wk. AC: anti-CFP immunouorescence staining in a representative ECFP/ANG II-transferred rat kidney. DF: anti-ANG II immunouorescence staining in a
representative ECFP/ANG II-transferred rat kidney. G: merged anti-CFP immunouorescence (blue-green) and nuclear DAPI staining (red) in a representative
control rat kidney without ECFP/ANG II transfer. H: merged anti-ANG II immunouorescence (green) and nuclear DAPI staining (red) in a representative control
rat kidney without ECFP/ANG II transfer. Bar 100 m.
Table 2. Relative time-dependent expression of ECFP/ANG II in the cortex and medulla of the rat kidney and extrarenal
tissues 7, 14, and 28 days after intrarenal adenoviral ECFP/ANG II transfer
ECFP/ANG II Expression, AFU
Structure Day 0 (n 8) Day 7 (n 12) Day 14 (n 16) Day 28 (n 11)
Glomerulus 12.9 4.3 18.9 6.3 26.0 6.5 21.2 3.4
Proximal tubule 26.1 5.6 156.6 28.6* 284.1 38.2 249.9 20.7
Cortical connecting tubule 10.2 2.8 13.6 4.6 22.0 6.1 28.3 5.2
Outer and inner medulla 5.5 1.3 7.5 2.3 13.5 4.6 11.3 3.8
Values are means SE expressed as arbitrary cyan uorescence units (AFU) quantied using the MetaMorph Imaging analysis system. ECFP, enhanced cyan
uorescence protein. Relative uorescence levels at day 0 represent those of background or autouorescence. Ectopic ECFP/ANG II expression was very low
to undetectable in extrarenal tissues including adrenals, brain, heart, liver, lung, and skeletal muscles. *P 0.01 vs. data obtained at day 0. P 0.01 vs. data
obtained at day 7.
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and mice in a time-dependent manner, with a peak response 2
wk after ECFP/ANG II transfer. The blood pressure-increasing
effect of ECFP/ANG II transfer was associated with signicant
increases in kidney and proximal tubule ANG II without
signicantly elevating plasma and urinary ANG II levels, and
with decreases in 24-h urine and fractional sodium and lithium
excretion, an indirect index of increased sodium reabsorption in
proximal tubules. Since the effects of ECFP/ANG II transfer were
blocked by concurrent losartan treatment in rats and prevented
in AT
1a
-KO mice, it is concluded that the AT
1
(AT
1a
) receptor
mediates the effects of this intracellular ANG II fusion protein
in the kidney.
One of major challenges to study the physiological role of
intracellular ANG II in the kidney is the difculty of admin-
istering ANG II into cells without rst binding to and activat-
ing cell surface AT
1
receptors. In the present study, we used a
novel ECFP-conjugated ANG II as an intracellular ANG II
protein, which was developed by Cook et al. (4, 28) using cyan
uorescent reporter proteins. There are unique advantages of
using this fusion protein for the current study: 1) it retains
biological activity of ANG II in COS-7 or CHO-K1 cells (4);
2) after its expression, the fusion protein is not secreted or
released into the medium (4); and 3) it allows direct visualiza-
tion and localization of the expressed ECFP/ANG II in the
tissues or target cells (3, 4). Indeed, the expression of ECFP/
ANG II in cultured mouse proximal tubule cells increased
intracellular ANG II levels by more than twofold, whereas the
levels of ANG II in the medium remained very low, thereby
conrming the results of Cook et al. (4) in COS-7 or CHO-K1
cells (4). More importantly in the present study, however, we
demonstrated that neither plasma nor urine ANG II levels or
CFP levels were signicantly increased by proximal tubule
cell-specic transfer of ECFP/ANG II in the rats. These results
indicate that the expressed ECFP/ANG II is not released or
secreted into the medium in vitro or into the renal interstitium
and proximal tubule lumen in vivo. Thus it is unlikely that
ECFP/ANG II was released or secreted into extracellular uid
compartments, bind and activate cell surface ANG II receptors
in the current study.
We have recently shown that microinjection of ANG II
directly into single proximal tubule cells increased intracellular
calcium (40), whereas in freshly isolated rat renal cortical
nuclei, ANG II induced in vitro transcription of transforming
growth factor-1, monocyte chemoattractant protein-1, and
sodium/hydrogen exchanger 3 (NHE-3) mRNAs (19). Both
effects were mediated by AT
1
(AT
1a
) receptors. It is not clear
whether these in vitro effects of intracellular ANG II can be
reproduced in proximal tubules of the kidney to induce a
physiological effect. Expression of an intracellular ANG II
selectively in proximal tubules of the kidney under the control
of a proximal tubule cell-specic promoter may be an ideal
approach. Keeping in this context, the kidney androgen-regu-
lated protein gene (KAP) has been used to drive proximal
tubule-specic expression of human angiotensinogen and renin
(7). The expression of the KAP gene is reportedly conned in
proximal tubule cells and regulated by androgen and estrogen
(7). Alternatively, the -glutamyl transpeptidase promoter was
used to knockin the AT
1a
receptor in proximal tubules of AT
1a
receptor-KO mice (13). These elegant approaches are very
useful for studying sexual dimorphic regulation of angio-
tensinogen expression (7) or the roles of AT
1a
receptors in
Fig. 7. Proximal tubule-selective expression of the scrambled version of
control fusion protein, ECFP/ANG IIc, in the rat kidney 2 wk after intrarenal
adenoviral transfer. A: ECFP/ANG II expression in the supercial cortex of a
representative ECFP/ANG IIc-transferred rat kidney (blue-green). B: represen-
tative isolated proximal tubule (PT) expressing ECFP/ANG IIc. C: represen-
tative isolated glomerulus lacking ECFP/ANG IIc expression. Bars 100 m
for the kidney section (A), 10 m for the isolated proximal tubule (B), or 30
m for the isolated glomerulus, respectively.
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proximal tubules (13), but they may not be suitable to deter-
mine the roles of intracellular vs. extracellular ANG II. In the
present study, we used the sglt2 promoter to drive ECFP/ANG
II expression selectively in proximal tubules of the rat and
mouse kidney. sglt2 is primarily expressed in the early S1
segment of proximal tubules (29), and the sglt2 gene promoter
was successfully used to drive the expression of the Cre
recombinase selectively in the proximal tubule to generate
specic Cre/Lox recombination in the mouse proximal tubule
(29). In the present study, we developed a recombinant human
adenoviral construct encoding the ECFP/ANG II plasmid and
the sglt2 gene promoter. Here, we demonstrated that intrarenal
adenoviral transfer of the ECFP/ANG II transgene via super-
cial cortical injection successfully transduces ECFP/ANG II
selectively in proximal tubules of rat kidneys (Figs. 37). The
expression of ECFP/ANG II is restricted primarily to the
proximal tubules in the cortex, and only very low levels of
ECFP/ANG II expression can be visualized in the glomeruli
(Figs. 37) or the entire medulla (Figs. 3 and 4). We found
negligible levels of ECFP/ANG II expression in the contralat-
eral, non-ECFP/ANG II-transferred kidney. Furthermore, we
did not observe signicant expression of ECFP/ANG II in all
extrarenal tissues examined (Fig. 8), suggesting that the ECFP/
ANG II plasmid or proteins had not spilled over into the
circulation and expressed in extrarenal tissues. Proximal tu-
bule-specic transfer of ECFP/ANG II is further conrmed by
increased ANG II levels in isolated proximal tubules but not in
plasma or urine (Fig. 9).
In a recent study, Redding et al. (28) created a unique
transgenic mouse strain expressing this particular intracellular
uorescent fusion of ANG II, which showed elevated blood
pressure and kidney pathology. The ECFP/ANG II protein is
expressed in all tissues including brain, heart, kidney, liver,
lung, and tests; thus the precise mechanisms responsible for
Fig. 8. Lack of ectopic expression of ECFP/ANG II in brain, liver, heart, adrenals, skeletal muscle, and lung 2 wk after intrarenal adenoviral transfer in rats.
Images shown are merged from cyan (ECFP/ANG II) and DAPI uorescence (nuclei). Bar 100 m.
Fig. 9. Effects of proximal tubule-specic
transfer of ECFP/ANG II on plasma, whole
kidney, isolated proximal tubule, and urinary
ANG II levels 2 wk after intrarenal transfer.
Note that ANG II levels were increased by
ECFP/ANG II expression in the kidney and
freshly isolated proximal tubules but not in
plasma and urine. *P 0.05 or **P 0.01 vs.
non-ECFP/ANG II-transferred control. P
0.05 or P 0.01 vs. ECFP/ANG II-
transferred.
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elevated blood pressure are not known. Nevertheless, this is the
rst transgenic mouse model with intracellular ANG II expres-
sion, which has demonstrated an effect on blood pressure in
animals. The current study extends this study by expressing
ECFP/ANG II selectively in proximal tubules of the rat kidney
using a proximal tubule-specic promoter, sglt2 (29). We
demonstrated that blood pressure was signicantly increased
by proximal tubule-specic transfer of ECFP/ANG II in a
time-related manner. The net increases in systolic blood pres-
sure 1, 2, or 4 wk after ECFP/ANG II transfer averaged
between 15 and 30 mmHg (Fig. 10). These levels of increased
blood pressure are similar to those reported in transgenic mice
globally expressing ECFP/ANG II (29) or in AT
1a
-KO mice
with the knockin of AT
1a
receptors selectively in proximal
tubules (13), but much lower than those induced by chronic
infusion of exogenous ANG II in rats or mice (15, 18, 24, 38).
The mechanisms by which intrarenal transfer of an intracel-
lular ANG II fusion protein selectively in proximal tubules of
the kidney increases blood pressure remain to be determined.
In cultured COS-7 or CHO-K1 cells, coexpression of ECFP/
ANG II with the AT
1
receptor fused to enhanced yellow
uorescent protein, AT
1
R/EYFP, signicantly induced cell
proliferation via activation of cAMP response element-associ-
ated protein (CREB) activity (4) or in A10 vascular smooth
muscle cells, ECFP/ANG II activated p38 MAP kinase (3).
Both effects were inhibited by losartan, suggesting an AT
1
receptor-dependent response. Baker et al. (1, 2, 12) demon-
strated that expression of an intracellular ANG II peptide in
cardiac myocytes in vitro or in vivo induced a hypertrophic
effect, a response that does not require activation of AT
1
receptors. Our present study strongly suggests that the blood
pressure-increasing effect of ECFP/ANG II expression selec-
tively in proximal tubules of the kidney is specic and depen-
dent on AT
1
(AT
1a
) receptor activation. Indeed, intrarenal
adenoviral transfer of a control scrambled sequence of ECFP/
ANG IIc selectively in proximal tubules using the same sglt2
promoter did not alter blood pressure in rats (Fig. 10). Further-
more, concurrent treatment of the rats with losartan completely
normalized systolic blood pressure elevated by proximal tu-
bule-specic transfer of ECFP/ANG II. Finally, the blood
pressure effect of ECFP/ANG II transfer was completely abol-
ished in AT
1a
receptor-KO mice (Fig. 10).
Although how ECFP/ANG II increases blood pressure
through activation of intracellular AT
1
(AT
1a
) receptors in
proximal tubules is not known, the present study suggests that
the effect may be in part mediated by increasing uid and
sodium reabsorption in proximal tubules via AT
1a
-mediated
transcriptional effect on NHE-3 expression. We have recently
shown that ANG II directly stimulated AT
1a
receptors to
induce transcriptional responses of NHE-3 in freshly isolated
rat renal cortical nuclei (19) and cultured proximal tubule cells
(14, 17). Furthermore, expression of ECFP/ANG II in cultured
mouse proximal tubule cells increases NHE-3 protein expres-
sion in wild-type, but not AT
1a
-KO, mouse proximal tubule
cells (Zhuo JL, Hopfer U, Li XL, unpublished observations).
Recent studies by Chappell and colleagues (8, 9, 25, 26)
suggest that reactive oxygen species (ROS) may be involved
Table 3. Peak effects of intrarenal adenoviral transfer of ECFP/ANG II or ECFP/ANG IIc selectively in proximal tubules of
the kidney on body and kidney weights, 24-h drinking, and urinary excretion of water and electrolytes in rats 14 days after
ECFP/ANG II transfer
Parameter Control (n 10) ECFP/ANG II (n 10) ECFP/ANG IILos (n 6) ECFP/ANG Iic (n 9)
Body wt, g 377 3 376 7 353 6 379 8
Left kidney wt 1.42 0.06 1.41 0.06 1.46 0.08 1.40 0.04
Left kidney wt-to-body wt ratio 100 0.38 0.05 0.38 0.07 0.41 0.08 0.36 0.04
Drinking, ml/24 h 40.1 1.6 43.6 2.3 42.4 1.64 40.5 1.2
V, ml/24 h 20.0 0.42 16.30 0.36* 21.84 0.55 18.2 0.3
UNaV, mmol/24 h 2.14 0.04 1.76 0.04* 2.33 0.06 2.10 0.02
UKV, mmol/24 h 4.97 0.06 4.16 0.07* 5.20 0.10 4.98 0.04
Values are means SE. Los, losartan; V, urine excretion; UNaV, urinary sodium excretion; UKV, urinary potassium excretion. There were no signicances
in these parameters between control and ECFP/ANG IIc-transferred rats. *P 0.05 vs. control. P 0.05 vs. ECFP/ANG II.
Fig. 10. Effects of proximal tubule-specic transfer of ECFP/ANG II or its
scrambled control, ECFP/ANG IIc, with or without losartan treatment on
systolic blood pressure in rats (SBP; A) or ECFP/ANG II transfer in wild-type
or AT1a-KO mice (B). *P 0.05 or **P 0.01 vs. basal SBP. P 0.05 or
P 0.01 vs. SBP in ECFP/ANG II-transferred rats. #P 0.05 or P
0.01 vs. SBP in wild-type mice at basal or 2 wk after intrarenal ECFP/ANG II
transfer.
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since ANG II appears to increase ROS in freshly isolated rat
and sheep renal cortical nuclei, which is also AT
1
receptor
dependent and may have long-term genomic effects. Increased
NHE-3 expression or activity in proximal tubules by intracel-
lular ANG II may lead to increases in proximal tubule sodium
transport and decreases in 24-h urinary sodium excretion. In the
present study, we carefully housed the animals individually in a
metabolic cage and provided the same ration of food and water
to maintain the same level of food and water intake for all rats.
Under these conditions, fractional sodium and lithium excre-
tion was signicantly decreased in rats transferred with ECFP/
ANG II at the peak expression level by day 14 (Table 2, Fig.
11). Lithium clearance has been used as an indirect index of
whole kidney proximal tubule sodium reabsorption (36, 37);
thus a decrease in fractional lithium excretion may be inter-
preted as an increase in proximal tubular sodium reabsorption.
Taken together, we suggest that proximal tubule-specic trans-
fer of ECFP/ANG II in rats or mice increases blood pressure
primarily via activation of AT
1
(AT
1a
) receptors to increase
proximal tubule sodium reabsorption in the kidney.
However, the present study may have some limitations. For
example, unlike the transgenic mouse model (29), our ap-
proach is adenovirus mediated, and the effects of ECFP/ANG
II transfer on blood pressure and proximal tubular function
unlikely last a lifetime. Second, we may not completely ex-
clude the possibility that ECFP/ANG II may interact with cell
surface receptors at the intracellular side. If this occurred,
ECFP/ANG II may activate cell surface receptors in a manner
similar to extracellular ANG II. In live cell uorescence
imaging studies, we did not visualize ECFP/ANG II secretion
into the medium or trafcked to the cell membranes. Third, to
increase the efciency or effectiveness of the gene transfer
selectively in proximal tubules of the kidney, blood ow to the
kidney would preferably be disrupted temporarily for several
minutes. This may lead to ischemic renal injury and therefore
elevate blood pressure. This seems unlikely since we con-
rmed that temporary disruption of renal blood ow for 5 min
did not cause apparent pathological changes in the kidneys of
control or ECFP/ANG II-transferred rats in preliminary stud-
ies. Other investigators have reported no irreversible kidney
injury with this gene transfer procedure (22, 23). Finally,
although at its peak intrarenal adenoviral transfer of this ANG
II fusion protein selectively in proximal tubules decreased
fractional sodium and lithium excretion while increasing blood
pressure, it may be difcult to infer that increased blood
pressure was directly due to sodium retention. Further studies
to simultaneously monitoring daily sodium balance and blood
pressure homeostasis in response to ECFP/ANG II transfer or
to directly study sodium transport responses in isolated prox-
imal tubule preparations may be necessary to determine the
cause and blood pressure effect relationship.
In summary, the present study demonstrates for the rst time
that recombinant human adenovirus and the sglt2 gene pro-
moter can effectively drive the expression of an intracellular
cyan uorescent fusion of ANG II protein selectively in prox-
Fig. 11. Effect of proximal tubule-specic transfer of ECFP/ANG II in the
kidney on fractional sodium (FENa) and lithium excretion (FELi) in ECFP/
ANG II-transferred rats 2 wk after the gene transfer. Fractional lithium
excretion was used as an indirect index of proximal tubular sodium reabsorp-
tion in the entire kidney (36, 37). A decrease in fractional lithium excretion
suggests an increase in overall proximal tubular sodium reabsorption in the
kidney. **P 0.01 vs. the control group without ECFP/ANG II transfer at day
14. P 0.01 vs. ECFP/ANG II-transferred rats.
Table 4. Long-term effects of proximal tubule-specic transfer of ECFP/ANG II in the rat kidney on body and left kidney
weights, systolic blood pressure, and 24-h urinary excretion of water and sodium 28 days after intrarenal ECFP/ANG II
transfer
Time Control (n 5) ECFP/ANG II (n 7)
Parameter Baseline Day 28 Baseline Day 28
Body wt, g 270 6 412 4* 269 6 412 6*
Left kidney wt, g 1.44 0.03 1.45 0.0
Heart wt, g 1.39 0.04 1.43 0.04
SBP, mmHg 118 4 126 8 119 4 146 9*
V, ml/24 h 11.7 0.6 17.6 0.6* 11.5 0.6 21.2 1.3*
UNaV, mmol/24 h 1.52 0.15 1.79 0.06* 1.34 0.14 2.19 0.04*
UkV, mmol/24 h 2.79 0.09 3.75 0.09* 2.99 0.11 4.07 0.09*
Values are means SE. SBP, systolic blood pressure. *P 0.01 vs. baseline in their respective group. P 0.05 vs. time control group at day 28.
Innovative Methodology
F1086 INTRACELLULAR ANGIOTENSIN II ON BLOOD PRESSURE
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imal tubules of the rat and mouse kidney in vivo. The ex-
pressed fusion protein is restricted primarily to proximal tu-
bules in the cortex and appears to remain intracellularly with-
out being released or secreted into the circulation or urine. No
apparent ectopic ECFP/ANG II expression was observed in
extrarenal tissues, thereby conrming the proximal tubule
cell-specic nature of the approach. The expression of ECFP/
ANG II in proximal tubules of one kidney appears to be
sufcient to elevate blood pressure, and the effect is likely
mediated by activation of AT
1
(AT
1a
) receptors and increases
in sodium and uid reabsorption in proximal tubules. Further
studies using isolated proximal tubule preparations or an in
vivo micropuncture technique in rats or mice with proximal
tubule-specic transfer of this intracellular ANG II fusion
protein may be necessary to further elucidate the physiological
effects and underlying cellular mechanisms of intracellular
ANG II on blood pressure regulation.
ACKNOWLEDGMENTS
Portions of this work were presented at the 63rd High Blood Pressure
Research Council Conference of the American Heart Association in Chicago,
IL, September 2326, 2009, and published as an abstract (Hypertension 54:
e73, 2009).
GRANTS
This work was supported in part by National Institutes of Health (NIH)
Grants 5RO1DK067299, 2R56DK067299, and 2RO1DK067299, an American
Society of Nephrology M. James Scherbenske Grant, and institutional support
from the Henry Ford Health System and the University of Mississippi Medical
Center to J. L. Zhuo. J. L. Cook was supported by NIH Grant R01 HL072795.
DISCLOSURES
No conicts of interest, nancial or otherwise, are declared by the authors.
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