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doi:10.1152/ajprenal.00329.2010 300:F1076-F1088, 2011. First published 9 February 2011;Am J Physiol Renal Physiol

ZhuoXiao C. Li, Julia L. Cook, Isabelle Rubera, Michel Tauc, Fan Zhang and Jia L.increases blood pressure in rats and micefusion of angiotensin II selectively in proximal tubules Intrarenal transfer of an intracellular fluorescent

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Intrarenal transfer of an intracellular fluorescent fusion of angiotensin IIselectively 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. Zhuo1,4

1Laboratory of Receptor and Signal Transduction, Department of Pharmacology and Toxicology, University of MississippiMedical Center, Jackson, Mississippi; 2Ochsner Clinic Foundation, New Orleans, Louisiana; 3UMR-CNRS 6548, Universityof Nice-Sophia Antipolis, Parc Valrose, Nice, France; and 4Division of Hypertension and Vascular Research, Departmentof Internal Medicine, Henry Ford Hospital, Detroit, Michigan

Submitted 8 June 2010; accepted in final form 3 February 2011

Li XC, Cook JL, Rubera I, Tauc M, Zhang F, Zhuo JL.Intrarenal transfer of an intracellular fluorescent fusion of angiotensinII selectively in proximal tubules increases blood pressure in rats andmice. Am J Physiol Renal Physiol 300: F1076–F1088, 2011. Firstpublished February 9, 2011; doi:10.1152/ajprenal.00329.2010.—Thepresent study tested the hypothesis that intrarenal adenoviral transferof an intracellular cyan fluorescent fusion of angiotensin II (ECFP/ANG II) selectively in proximal tubules of the kidney increases bloodpressure by activating AT1 (AT1a) receptors. Intrarenal transfer ofECFP/ANG II was induced in the superficial cortex of rat and mousekidneys, and the sodium and glucose cotransporter 2 (sglt2) promoterwas used to drive ECFP/ANG II expression selectively in proximaltubules. Intrarenal transfer of ECFP/ANG II induced a time-depen-dent, proximal tubule-selective expression of ECFP/ANG II in thecortex, which peaked at 2 wk and was sustained for 4 wk. ECFP/ANGII expression was low in the glomeruli and the entire medulla and wasabsent in the contralateral kidney or extrarenal tissues. At its peak ofexpression in proximal tubules at day 14, ANG II was increased bytwofold in the kidney (P � 0.01) and more than threefold in proximaltubules (P � 0.01), but remained unchanged in plasma or urine.Systolic blood pressure was increased in ECFP/ANG II-transferredrats by 28 � 6 mmHg (P � 0.01), whereas fractional sodiumexcretion was decreased by 20% (P � 0.01) and fractional lithiumexcretion was reduced by 24% (P � 0.01). These effects were blockedby losartan and prevented in AT1a knockout mice. Transfer of ascrambled ECFP/ANG IIc had no effects on blood pressure, kidney,and proximal tubule ANG II, or sodium excretion. These resultsprovide evidence that proximal tubule-selective transfer of an intra-cellular ANG II fusion protein increases blood pressure by activatingAT1a receptors and increasing sodium reabsorption in proximal tu-bules.

adenoviral gene delivery; G protein-coupled receptors; intracrinepeptides; losartan; renin-angiotensin-aldosterone system; urinary so-dium excretion

THE RENIN-ANGIOTENSIN SYSTEM (RAS) is long recognized to existand function as dual extracellular (endocrine and/or paracrine)and intracellular (or intracrine) vasoactive hormonal systems.The extracellular system includes circulating and local tissueANG II, which plays the classic roles of ANG II throughactivation of cell surface G protein-coupled ANG II receptors(GPCRs) (6, 21, 30, 35). The intracellular system includesintracellularly formed ANG II (1–5, 8–10, 12, 19, 40) andextracellular ANG II internalized through type 1 (AT1) recep-tor-mediated endocytosis (14, 15, 18, 19, 25, 27, 39). The rolesof 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). Bycontrast, whether intracellular ANG II may induce any physi-ological effects remains largely unknown. The slow progress inour understanding of physiological roles of intracellular ANGII has been stymied in part due to the lack of suitable animalmodels that express an ANG II peptide in a particular tissue orcell, which is produced intracellularly but not released orsecreted into the extracellular fluid compartment or the circu-lation, and the technical challenges in distinguishing betweenthe effects induced by intracellular vs. extracellular ANG IIthrough activation of its intracellular vs. cell surface receptors.

Transgenic mice have been generated to express an ANGII-producing fusion protein in cardiomyocytes using the �-my-osin heavy chain promoter (32, 34). This cardiac-specific ANGII construct contains a signal peptide sequence from humanprorenin and a furin cleavage site. ANG II fusion protein canbe cleaved by furin, released into the secretory pathway, andsecreted into the cardiac interstitium (32, 34). Thus this ANGII fusion protein activates cell surface rather than intracellularor nuclear receptors to induce paracrine effects similar toextracellular ANG II. Alternatively, an adenoviral vector en-coding an intracellular ANG II peptide has been used to studythe hypertrophic effect of cardiac-specific ANG II (1). Intra-cardiac transduction of this peptide in mice resulted in cardiachypertrophy without affecting blood pressure and circulatingANG II levels. However, the cardiac hypertrophic effect in-duced by this intracellular ANG II is not blocked by the AT1

receptor blocker losartan, suggesting an AT1 receptor-indepen-dent effect of this novel intracellular ANG II peptide. Mostrecently, transgenic mice expressing an intracellular fluores-cent fusion of ANG II have been generated using the mousemetallothionein promoter (28). Global expression of this trans-gene, enhanced cyan fluorescent protein (ECFP)/ANG II, in alltissues, 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 constructencoding a fluorescent 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 mannerthat ensures ECFP/ANG II to be synthesized on free ribo-somes, but not destined to the secretory pathway for secretionout of the cells (4, 28). The sodium and glucose cotransporter2 (sglt2) promoter was used to drive the expression of ECFP/ANG II selectively in proximal tubules of rat and mousekidneys (29). We report here for the first time that intrarenaladenoviral transfer of this intracellular ANG II fusion protein

Address for reprint requests and other correspondence: J. L. Zhuo, Dept. ofPharmacology and Toxicology, Univ. of Mississippi Medical Center, 2500North State St., Jackson, MS 39216-4505 (e-mail: [email protected]).

Am J Physiol Renal Physiol 300: F1076–F1088, 2011.First published February 9, 2011; doi:10.1152/ajprenal.00329.2010.Innovative Methodology

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selectively in proximal tubules of rat and mouse kidneys canincrease blood pressure and decreased fractional sodium andlithium excretion through AT1a receptor-dependent mecha-nisms.

METHODS

Construction of the ECFP/ANG II transgene. The expression plas-mid encoding a cyan fluorescent fusion of ANG II, ECFP/ANG II, anda scrambled version of the plasmid were generated by Dr. Julie Cookof the Ochsner Clinic Foundation. Construction of ECFP/ANG II orits scrambled version, ECFP/ANG IIc, was previously described (4).Briefly, ANG II or ANG IIc was ligated downstream of ECFP in thecyan fluorescent fusion vector pECFP-C1 (Clontech, Palo Alto, CA) togenerate a cyan fluorescent fusion protein, ECFP/ANG II or ECFP/ANGIIc. 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-digestedpECFP-C1. For ECFP/ANG IIc, the upstream primer was 5=-AGCTTCATACGACCACCGCGTATTTCCCATCTAGG-3=, and thedownstream primer was 5=-GATCCCTAGATGGGAAATACGCG-GTGGTCGTATGA-3= (4). The cloned plasmids were further sequencedfor confirmation.

Construction of the ECFP/ANG II transgene with the proximaltubule-specific promoter sglt2. The sglt2 promoter was provided byDrs. Isabelle Rubera and Michel Tauc of the University of Nice-Sophia Antipolis (Nice, France) and was used to drive the expressionof ECFP/ANG II or ECFP/ANG IIc selectively in proximal tubules ofthe kidney (29). The promoter contains 2,637 bp of the murine sglt25=-flanking region (nucleotides 55-2691 of GenBank accession no.AJ292928, exon 1 within the initiation codon ATG, intron 1, and thefirst part 923 bp of exon 2) (24). To enable proximal tubule-specificexpression of ECFP/ANG II or ECFP/ANG IIc using the sglt2promoter, the NotI fragment (2.6 kb) of pGEM-sglt2–5pr-mut wasfirst subcloned into the NotI-digested DUAL-Basic vector. The NheI/AflII/Klenow fragment (1.1 kb) of pECFP/ANG II was then subclonedinto XhoI/Klenow-digested DUAL-sglt2–5pr-mut (Vector BioLab,Philadelphia, PA). The entire expression cassette was then transferredto the adenovirus genome vector and confirmed through restrictionmapping with a titer of �4.6 � 1011 pfu/ml. The specific adenoviralvector used in this study was Ad-sglt2-ECFP/ANG II, and the controlvector was Ad-sglt2-ECFP/ANG IIc (Fig. 1). The specificity of thesglt2 promoter to drive specific expression of Cre recombinase in thekidney proximal tubules has been confirmed in a transgenic Cre/Loxmouse model (29).

Expression of ECFP/AII in cultured mouse proximal tubule cells.Semiconfluent mouse proximal tubule cells (a gift from Dr. UlrichHopfer, Case Western Reserve University) in six-well plates or onglass coverslips were transfected with the specific 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 describedpreviously (14, 17, 40). The medium was collected for measurementof ECFP as a marker of expression using a fluorescent plate reader,whereas the cells were visualized using a Nikon-Eclipse TE2000-Uinverted fluorescence microscope and a dual 4=,6-diamidino-2-phe-nylindole (DAPI)-CFP filter set (excitation: 440 nm; emission: 480nm). In further experiments, ANG II peptides were extracted from themedium and proximal tubule cells for measurements of ANG II levelsas we described previously (14, 17, 19, 40).

Intrarenal adenoviral transfer of ECFP/ANG II or ECFP/ANG IIc.Seven groups (n � 6–16 each) of adult male Sprague-Dawley rats (74in total) and two groups (n � 8 each) of wild-type (C57BL/6J; 16 intotal) and AT1a receptor-deficient mice (Agtr1a�/� or AT1a-KO; 16in total) were used in the current study. All animals were maintainedon a normal ration of rodent chow and had free access to tap water.Basal systolic blood pressure (SBP), 24-h drinking, urine, and urinarysodium excretion were first determined before intrarenal transfer ofECFP/ANG II or ECFP/ANG IIc was performed. To induce intrarenaladenoviral ECFP/ANG II transfer, rats and wild-type or AT1a-KOmice were anesthetized and their left renal artery was temporarilyclamped with a fine vessel clip, which briefly interrupted blood flowto 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 anddirectly injected into the superficial cortex evenly with six locations(20 �l each) (22, 23). By contrast, the control groups of rats or micereceived sham injections of saline instead. Blood flow to the leftkidney was reestablished 5 min after injection of Ad-sglt2-ECFP/ANG II or Ad-sglt2-ECFP/ANG IIc. The duration of renal blood flowdisruption for a brief 5 min was chosen and based on a series ofpreliminary time-dependent studies (5–15 min). No long-term histo-logical or pathological changes were identified while effective intrare-nal adenoviral gene transfer with renal blood flow interruption for 5min was ensured. Similar findings were reported previously in rats(22, 23). Animals were then maintained for 1 wk or up to 4 wkdependent on a particular experimental protocol. To determine therole of AT1 receptors in mediating the effects of ECFP/ANG IItransfer in rats, a group of rats was transferred with ECFP/ANG II inproximal tubules and concurrently treated with the AT1 receptorblocker losartan (20 mg·kg�1·day�1 po) for 2 wk. All experimentsusing animals were approved by the Institutional Animal Care andUse and Recombinant DNA and Biosafety Committees of the HenryFord Health System (Detroit, MI) and the University of MississippiMedical Center (Jackson, MS), respectively.

Measurement of blood pressure and 24-h drinking and urinaryexcretion of water and electrolytes. Basal and weekly systolic bloodpressure in rats and mice was measured for 1–4 wk using the tail-cuffmethod as we described previously (15, 16, 18, 38). Basal and weekly24-h drinking, urine, and urinary sodium and potassium excretionwere measured using a metabolic cage and a NOVA 13 electrolyteanalyzer (Nova Biomedicals) in all animals maintained on the samerations of chow and water as we described previously for 1–4 wk (15,16, 18, 38).

Measurement of plasma and urine creatinine and lithium concentrations.Plasma and urine creatinine concentrations were determined using acreatinine parameter colorimetric assay kit (R&D Systems, Minneap-olis, MN) (11, 20). To measure lithium clearance as an indirect indexof proximal tubule sodium reabsorption in the rat kidney, animalswere fed a chow containing 15 mmol/kg dry wt food throughout theexperiment as described elsewhere (36, 37). Lithium concentrationswere measured using a NOVA 13 electrolyte analyzer (Nova Bio-medicals). Creatinine and lithium clearance were calculated using thestandard renal clearance method (11, 20, 36, 37).

Measurements of plasma, kidney, proximal tubule, and urine ANGII levels. At the end of experiment, rats were decapitated withoutanesthesia, and trunk blood samples collected to measure plasmaECFP and ANG II levels as described (15, 18, 24, 38). The left kidneywas sliced through the middle into two portions, with one-half

Fig. 1. Schematic construction map of recombinant human adenoviral vectorscarrying the cyan fluorescent fusion of ANG II (ECFP/ANG II) or itsscrambled control ANG IIc (ECFP/ANG IIc) and the sodium and glucosecotransporter 2 (sglt2) gene promoter.

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immediately processed for measurement of the whole kidney ANG IIlevels as described (15, 18, 24, 38). The other half of the left kidneywas used to isolate fresh proximal tubules from the superficial cortexby collagenase digestion, sequential filtrations, and separation on a50% Percoll gradient (33). In separate experiments, five rat superficialcortex samples were dissected on ice into three portions to determinethe rate of ANG II degradation due to collagenase digestion, filtration,and separation of proximal tubules. The first portion was immediatelyprocessed for measuring renal cortical ANG II levels without beingprocessed through collagenase digestion and sequential separationprocedures. ANG II levels measured as such were taken as an indirectindex of proximal tubule ANG II without degradation, because prox-imal tubules account for 90% of tissues in the superficial cortex. Thesecond portion was digested with collagenase and left at 37°C in abuffer containing an inhibitor cocktail for �45 min, the time requiredfor the isolation of proximal tubules. The differences in ANG II levelsbetween the first and second portions of cortical samples represent theANG II levels degraded during the proximal tubule isolation pro-cesses. The third portion of samples was used for collagenase diges-tion at 37°C and sequential separation of proximal tubules in a buffercontaining an inhibitor cocktail. ANG II levels measured as suchrepresent intracellular ANG II levels in proximal tubules followingcollagenase digestion and degradation (Table 1). Plasma, kidney, andproximal tubule ANG II were measured using a sensitive ELISA kit(Bachem) (14, 15, 17, 18). Proximal tubule ANG II levels werecorrected by the 42% of ANG II degradation rate during collagenasedigestion and sequential separation of proximal tubules. In a recentstudy, van Esch et al. (31) showed that close to 70% of ANG II in thekidney was metabolized if the samples were simply left at roomtemperature. ANG II was also extracted from urine samples using aphenyl-bonded solid-phase peptide extraction column (Elut-C18, Var-ian), vacuum-dried overnight, and reconstituted in an ANG II assaybuffer. Urinary ANG II was measured as described for plasma ANGII and corrected by 24-h urine excretion.

Fluorescent microscopic imaging of ECFP/ANG II or ECFP/ANGIIc expression in the kidney and extrarenal tissues. To confirm theproximal tubule-specific expression and to exclude the ectopic expres-sion of ECFP/ANG II or ECFP/ANG IIc in the kidney and/orextrarenal tissues, fresh and frozen sections (6-�m thick) were cut ona cryostat and thaw-mounted on glass slides from adrenal glands,brain, heart, kidney, liver, lung, and skeletal smooth muscles ofseparate groups of rats with or without intrarenal ECFP/ANG II orECFP/ANG IIc transfer. Sections were then briefly counterstainedwith the cell nuclear marker DAPI (300 nM) for 5 min, washed withphosphate-buffered saline, and mounted on a microscopic stage.Expression of ECFP/ANG II or ECFP/ANG IIc in these tissues wasvisualized using a Nikon-Eclipse TE2000-U inverted fluorescencemicroscope and a dual DAPI-CFP band-pass excitation filter set(excitation: 440 nm; emission: 495/50 nm). Nuclear DAPI-stainedimages were converted into red for better differentiation betweenECFP (blue-green) and DAPI (blue). In all fluorescence imaging

analyses, the background autofluorescence level was determined inthe renal medulla of the same kidney with ECFP/ANG II or ECFP/ANG IIc transfer in the superficial cortex or in the contralateral kidneythat was not transferred with ECFP/ANG II or ECFP/ANG IIc.

Fluorescent immunohistochemistry of CFP and ANG II in thekidney. To provide additional evidence for the increased expression ofECFP and ANG II specifically in proximal tubules of the kidney,fluorescent immunohistochemistry was performed using a mousemonoclonal anti-cyan fluorescence protein (CFP) antibody (1:250,Abm, Richmond, ON) or a rabbit anti-human ANG II antibody (1:250,USBiological, Swampscott, MA), respectively. Secondary antibodiesused 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 visualizedusing a Nikon-Eclipse TE2000-U inverted fluorescence microscopeand a dual DAPI-FITC band-pass excitation filter set (excitation: 488nm; emission: 510/50 nm). For better image visualization or differ-entiation, nuclear DAPI staining was converted to red, ANG IIimmunostaining to green, and CFP immunostaining to blue-greenimages, respectively.

Statistical analysis. All results are presented as means � SE.One-way ANOVA was first to compare the differences in the sameparameters between groups of rats or mice. If the P value was �0.05,a post hoc Newman-Keuls multiple comparison test was performedto compare two different group means. The significance was set atP � 0.05.

RESULTS

Expression of ECFP/ANG II in cultured proximal tubulecells. Figure 2 shows that transfection of mouse proximaltubule cells with the adenoviral construct Ad-sglt2-ECFP/ANGII (4 �g/well) for 48 h induced intensive expression of ECFP/ANG II throughout the cytoplasm and perinuclear regions ofthe cells. Expression of ECFP/ANG II increased intracellularANG 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 tubuleANG II levels (246.4 � 37.9 pg/mg protein, not significant vs.control). By contrast, ANG II levels in the medium remainedvery low (26.1 � 3.8 pg/mg protein). There were no significantdifferences in the medium ECFP levels between control cellsand cells transfected with either ECFP/ANG II or ECFP/ANGIIc (not shown).

Proximal tubule-specific expression of ECFP/ANG II in thekidney. Intrarenal adenoviral transfer of ECFP/ANG II usingthe sglt2 promoter led to a time-dependent expression of thetransgene (cyan fluorescence as blue-green) selectively inproximal tubules throughout the superficial cortex (Fig. 3,A–I). Close visualization of serial longitudinal sections of thekidneys that were transferred with ECFP/ANG II (n � 6 foreach time point) showed that the expression of ECFP/ANG IIreached 78 � 6% of proximal tubules in the cortex 7 days afterintrarenal transfer (Fig. 3A). The proximal tubule-specific ex-pression of ECFP/ANG II was increased further to 92 � 3% by2 wk (Fig. 3D) and persisted for 4 wk after transfer (Fig. 3G).There were very low levels of ECFP/ANG II expression in theglomeruli (Fig. 3, A, D, and G) or in the outer renal medulla inthe same kidney that was transferred with ECFP/ANG II in thesuperficial cortex (Fig. 3, J–R). Figure 4 compares ECFP/ANGII expression between the left kidney with the ECFP/ANG IItransfer and the contralateral right kidney without ECFP/ANGII transfer. While ECFP/ANG II expression was observedselectively in all proximal tubules of the left kidney (Fig. 4A),

Table 1. Effects of collagenase digestion and proximaltubule isolation procedures on ANG II degradation ormetabolism in the rat kidney

Superficial Cortex(0°C)

Superficial Cortex(37°C)

Isolated ProximalTubule (37°C)

ANG II, pg/mg protein 129.2 � 11.3 75.6 � 6.8* 78.0 � 14.5*

Values are means � SE. Superficial cortical samples was taken as anindirect index of proximal tubule samples, because proximal tubules accountfor 90% of tissues in the superficial cortex. Note that ANG II level wasdecreased by 42% in superficial cortical samples subjected to collagenasedigestion and proximal tubule isolation buffer at 37°C. *P � 0.01 vs.superficial cortical samples immediately homogenized in an ANG II extractionbuffer on ice and extracted for ANG II assays (0°C) (15, 18, 38).


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no significant ECFP/AII expression was seen in proximaltubules of the contralateral right kidney (Fig. 4C). Similarly,very low levels of ECFP/ANG II expression were expressed inthe inner medulla of left (Fig. 4B) and right kidneys (Fig. 4D).Furthermore, ECFP/AII expression was seen throughout thetubule wall in freshly isolated proximal tubules (Fig. 5, A andC), whereas no significant ECFP/ANG II expression wasobserved in freshly isolated glomeruli (Fig. 5, D and F).

To further confirm the increased expression of ECFP andANG II in proximal tubules of the rat kidney with ECFP/ANGII transfer, fluorescent immunohistochemistry was performedusing a specific anti-CFP or anti-ANG II antibody (Fig. 6).Increased anti-CFP (blue-green, Fig. 6, A and C) and anti-ANGII (green, Fig. 6, D and F) immunofluorescence staining wasobserved in all proximal tubules of the kidneys with ECFP/ANG II transfer. In the control rat kidney without ECFP/ANGII transfer, little anti-CFP immunofluorescence staining wasobserved in the glomeruli and proximal tubules (Fig. 6G).However, anti-ANG II immunostaining was seen in proximaltubules of the control kidney, but the level was much lowerthan that found in the ECFP/ANG II-transferred kidney (Fig. 6,D and F). The relative time-related expression of ECFP/ANGII in the glomeruli, proximal tubules, and the medulla of thekidney is summarized in Table 2.

For a direct comparison, proximal tubule-specific expres-sion of the scrambled version of the fusion protein ECFP/ANG IIc in the rat kidney is shown in Fig. 7. The pattern ofECFP/ANG IIc expression or its distribution in proximaltubules 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 IIescapes into the circulation and induces ectopic expression inother extrarenal tissues, the heart, liver, spleen, brain, lung, andadrenals were examined for ECFP/ANG II expression. Figure 8

shows that intrarenal adenoviral transfer of ECFP/ANG IIusing the sglt2 promoter did not lead to significant ectopicexpression of the transgene in all extrarenal tissues we exam-ined.

Effects of proximal tubule-specific transfer of ECFP/ANG IIon plasma, kidney, proximal tubule, and urine ANG II levels.As shown in Fig. 9A, plasma ANG II levels were not differentbetween control and ECFP/ANG II-transferred rats (control:228.9 � 30.9 vs. ECFP/ANG II: 247.6 � 36 fmol/ml, notsignificant). By contrast, ANG II levels in the kidney wereincreased significantly by more than twofold in rats 2 wk afterintrarenal 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 morethan threefold from 73.8 � 10.2 pg/mg protein in control ratsto 252.7 � 13.2 pg/mg protein in ECFP/ANG II-transferredrats (P � 0.01) (Fig. 9C). Concurrent treatment with losartan inECFP/AII-transferred rats significantly reduced kidney ANG II(643.2 � 48.0 pg/g kidney wt, P � 0.05) and proximal tubuleANG II (115.9 � 12.3 pg/mg protein, P � 0.01) to levels lowerthan those of ECFP/ANG II-transferred but higher than thoseof control rats. By contrast, plasma ANG II levels in ECFP/ANG II-transferred rats treated with losartan were increased byabout threefold over control rats in part due to inhibition ofAT1 receptor-mediated uptake of circulating ANG II in tissues(ECFP/ANG IIlosartan: 705.1 � pg/ml, P � 0.01). Therewere no significant differences in 24-h urinary ANG II excre-tion between the ECFP/ANG II-transferred and nontransferredrats (control: 466.8 � 46.3 vs. ECFP/ANG II: 492.1 � 36.5pg/24 h, not significant) (Fig. 9D).

Intrarenal adenoviral transfer of the scrambled version of thefusion protein ECFP/ANG IIc had no significant effects onplasma, kidney, proximal tubule, and urinary ANG II levelscompared with control rats (Fig. 9).

Fig. 2. Expression of an intracellular cyan flu-orescent fusion of ANG II, ECFP/ANG II, incultured mouse proximal tubule cells 48 h aftertransfection. A: expression of ECFP/ANG II inthe cytoplasm and perinuclear region, shown asblue-green. B: 4=,6-diamidino-2-phenylindole(DAPI)-stained nuclei, which was converted tored to facilitate visualization. C: merged imageof 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-specific transfer of ECFP/ANG IIselectively in proximal tubules on systolic blood pressure inrats. Baseline systolic blood pressure was similar in all groupsof animals before ECFP/ANG II transfer was performed,which remained unchanged throughout the study in shamcontrol rats (Fig. 10A). In rats with intrarenal transfer ofECFP/ANG II selectively in proximal tubules, systolic bloodpressure increased from a baseline of 118 � 4 to 137 � 4mmHg at day 7 (P � 0.01) and further to 149 � 3 mmHg atday 14 after ECFP/ANG II transfer (P � 0.01). Concurrent

treatment with losartan in rats treated with ECFP/ANG IItransfer prevented the increases in blood pressure induced byECFP/ANG II (Fig. 10A). Intrarenal adenoviral transfer of thecontrol 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 andthen weekly for 4 wk. Systolic blood pressure remained ele-vated 4 wk after ECFP/ANG II transfer in the kidney (seeTable 4).

Fig. 3. Time-dependent expression of ECFP/ANG II in proximal tubules of the rat renalcortex (A–I), but not in the outer medulla(J–R), 7, 14, or 28 days after intrarenal ad-enoviral transfer of ECFP/ANG II in thesuperficial 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 DAPIstaining, which was converted to red to fa-cilitate visualization. C, F, I, L, O, andR: merged or overlaid images of ECFP/ANGII and DAPI staining. G, glomerulus; PT,proximal tubule. Bars � �100 �m.




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Effect of proximal tubule-specific transfer of ECFP/ANG IIon blood pressure in AT1a-KO mice. In wild-type C57BL/6Jmice, intrarenal adenoviral ECFP/ANG II transfer significantlyincreased systolic blood pressure at day 14 (control: 122 � 2vs. ECFP/ANG II: 154 � 3 mmHg, P � 0.01) (Fig. 10B).However, intrarenal adenoviral ECFP/ANG II transfer had nosignificant effect on blood pressure in AT1a-KO mice (control:98 � 3 vs. ECFP/ANG II: 102 � 8 mmHg, not significant)(Fig. 10B).

Effects of proximal tubule-specific transfer of ECFP/ANG IIor ECFP/ANG IIc on body, heart, and kidney weights and 24-hurine excretion and fractional sodium and lithium excretion.Proximal tubule expression of ECFP/ANG II or ECFP/ANGIIc did not significantly 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 proximaltubules decreased 24-h urine and urinary sodium and potas-sium excretion 2 wk after ECFP/ANG II transfer, and theresponses 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 significantly decreased by intrarenal ECFP/ANG IItransfer. 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 afterproximal tubule-specific 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 wkafter intrarenal adenoviral transfer. A: specificECFP/ANG II expression in proximal tubulesof the left kidney, which received ECFP/ANGII transfer in the superficial cortex. B: lack ofECFP/ANG II expression in the left inner me-dulla. C: lack of ECFP/ANG II expression inthe right contralateral renal cortex. D: lack ofECFP/ANG II expression in the right contralat-eral inner medulla. Images shown are mergedfrom cyan (ECFP/ANG II) and DAPI fluores-cence (nuclei). G, glomerulus. PT, proximaltubules. Bar � �100 �m.




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urinary excretion of water and sodium returned to the levelssignificantly higher than those of time control rats 28 days afterECFP/ANG II transfer in the presence of continuously elevatedblood pressure (Table 4).

DISCUSSION

Although intracellularly administered ANG II can inducebiological effects in cultured cells or in freshly isolated nuclei(2, 4, 8–10, 19, 25, 26), whether intracellular ANG II plays a

physiological role in the regulation of blood pressure and renalfunction is unknown. The goal of the current study was to testthe hypothesis that intrarenal adenovirus-mediated transfer ofan intracellular cyan fluorescent fusion of ANG II (ECFP/ANGII) selectively in proximal tubules of the rat and mouse kidneysincreases arterial blood pressure by activating AT1 (AT1a)receptors in the kidney. Our current results demonstrate thatproximal tubule cell-specific transfer of this intracellular ANGII fusion protein indeed increases systolic blood pressure in rats

Fig. 6. Immunofluorescence staining for increased expression of ECFP and ANG II in proximal tubules of the rat kidney transferred with ECFP/ANG II for 2wk. A–C: anti-CFP immunofluorescence staining in a representative ECFP/ANG II-transferred rat kidney. D–F: anti-ANG II immunofluorescence staining in arepresentative ECFP/ANG II-transferred rat kidney. G: merged anti-CFP immunofluorescence (blue-green) and nuclear DAPI staining (red) in a representativecontrol rat kidney without ECFP/ANG II transfer. H: merged anti-ANG II immunofluorescence (green) and nuclear DAPI staining (red) in a representative controlrat 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 extrarenaltissues 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 � 3.4Proximal 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 fluorescence units (AFU) quantified using the MetaMorph Imaging analysis system. ECFP, enhanced cyanfluorescence protein. Relative fluorescence levels at day 0 represent those of background or autofluorescence. Ectopic ECFP/ANG II expression was very lowto 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. dataobtained at day 7.




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and mice in a time-dependent manner, with a peak response 2wk after ECFP/ANG II transfer. The blood pressure-increasingeffect of ECFP/ANG II transfer was associated with significantincreases in kidney and proximal tubule ANG II withoutsignificantly elevating plasma and urinary ANG II levels, andwith decreases in 24-h urine and fractional sodium and lithiumexcretion, an indirect index of increased sodium reabsorption inproximal tubules. Since the effects of ECFP/ANG II transfer wereblocked by concurrent losartan treatment in rats and preventedin AT1a-KO mice, it is concluded that the AT1 (AT1a) receptormediates the effects of this intracellular ANG II fusion proteinin the kidney.

One of major challenges to study the physiological role ofintracellular ANG II in the kidney is the difficulty of admin-istering ANG II into cells without first binding to and activat-ing cell surface AT1 receptors. In the present study, we used anovel ECFP-conjugated ANG II as an intracellular ANG IIprotein, which was developed by Cook et al. (4, 28) using cyanfluorescent reporter proteins. There are unique advantages ofusing this fusion protein for the current study: 1) it retainsbiological activity of ANG II in COS-7 or CHO-K1 cells (4);2) after its expression, the fusion protein is not secreted orreleased into the medium (4); and 3) it allows direct visualiza-tion and localization of the expressed ECFP/ANG II in thetissues or target cells (3, 4). Indeed, the expression of ECFP/ANG II in cultured mouse proximal tubule cells increasedintracellular ANG II levels by more than twofold, whereas thelevels of ANG II in the medium remained very low, therebyconfirming the results of Cook et al. (4) in COS-7 or CHO-K1cells (4). More importantly in the present study, however, wedemonstrated that neither plasma nor urine ANG II levels orCFP levels were significantly increased by proximal tubulecell-specific transfer of ECFP/ANG II in the rats. These resultsindicate that the expressed ECFP/ANG II is not released orsecreted into the medium in vitro or into the renal interstitiumand proximal tubule lumen in vivo. Thus it is unlikely thatECFP/ANG II was released or secreted into extracellular fluidcompartments, bind and activate cell surface ANG II receptorsin the current study.

We have recently shown that microinjection of ANG IIdirectly into single proximal tubule cells increased intracellularcalcium (40), whereas in freshly isolated rat renal corticalnuclei, ANG II induced in vitro transcription of transforminggrowth factor-�1, monocyte chemoattractant protein-1, andsodium/hydrogen exchanger 3 (NHE-3) mRNAs (19). Botheffects were mediated by AT1 (AT1a) receptors. It is not clearwhether these in vitro effects of intracellular ANG II can bereproduced in proximal tubules of the kidney to induce aphysiological effect. Expression of an intracellular ANG IIselectively in proximal tubules of the kidney under the controlof a proximal tubule cell-specific promoter may be an idealapproach. Keeping in this context, the kidney androgen-regu-lated protein gene (KAP) has been used to drive proximaltubule-specific expression of human angiotensinogen and renin(7). The expression of the KAP gene is reportedly confined inproximal tubule cells and regulated by androgen and estrogen(7). Alternatively, the �-glutamyl transpeptidase promoter wasused to knockin the AT1a receptor in proximal tubules of AT1a

receptor-KO mice (13). These elegant approaches are veryuseful for studying sexual dimorphic regulation of angio-tensinogen expression (7) or the roles of AT1a receptors in

Fig. 7. Proximal tubule-selective expression of the scrambled version ofcontrol fusion protein, ECFP/ANG IIc, in the rat kidney 2 wk after intrarenaladenoviral transfer. A: ECFP/ANG II expression in the superficial cortex of arepresentative 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 �mfor 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 thepresent study, we used the sglt2 promoter to drive ECFP/ANGII expression selectively in proximal tubules of the rat andmouse kidney. sglt2 is primarily expressed in the early S1segment of proximal tubules (29), and the sglt2 gene promoterwas successfully used to drive the expression of the Crerecombinase selectively in the proximal tubule to generatespecific Cre/Lox recombination in the mouse proximal tubule(29). In the present study, we developed a recombinant humanadenoviral construct encoding the ECFP/ANG II plasmid andthe sglt2 gene promoter. Here, we demonstrated that intrarenaladenoviral transfer of the ECFP/ANG II transgene via super-ficial cortical injection successfully transduces ECFP/ANG IIselectively in proximal tubules of rat kidneys (Figs. 3–7). Theexpression of ECFP/ANG II is restricted primarily to theproximal tubules in the cortex, and only very low levels of

ECFP/ANG II expression can be visualized in the glomeruli(Figs. 3–7) or the entire medulla (Figs. 3 and 4). We foundnegligible levels of ECFP/ANG II expression in the contralat-eral, non-ECFP/ANG II-transferred kidney. Furthermore, wedid not observe significant expression of ECFP/ANG II in allextrarenal tissues examined (Fig. 8), suggesting that the ECFP/ANG II plasmid or proteins had not spilled over into thecirculation and expressed in extrarenal tissues. Proximal tu-bule-specific transfer of ECFP/ANG II is further confirmed byincreased ANG II levels in isolated proximal tubules but not inplasma or urine (Fig. 9).

In a recent study, Redding et al. (28) created a uniquetransgenic mouse strain expressing this particular intracellularfluorescent fusion of ANG II, which showed elevated bloodpressure and kidney pathology. The ECFP/ANG II protein isexpressed 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 fluorescence (nuclei). Bar � 100 �m.

Fig. 9. Effects of proximal tubule-specifictransfer of ECFP/ANG II on plasma, wholekidney, isolated proximal tubule, and urinaryANG II levels 2 wk after intrarenal transfer.Note that ANG II levels were increased byECFP/ANG II expression in the kidney andfreshly isolated proximal tubules but not inplasma 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 thefirst transgenic mouse model with intracellular ANG II expres-sion, which has demonstrated an effect on blood pressure inanimals. The current study extends this study by expressingECFP/ANG II selectively in proximal tubules of the rat kidneyusing a proximal tubule-specific promoter, sglt2 (29). Wedemonstrated that blood pressure was significantly increasedby proximal tubule-specific transfer of ECFP/ANG II in atime-related manner. The net increases in systolic blood pres-sure 1, 2, or 4 wk after ECFP/ANG II transfer averagedbetween 15 and 30 mmHg (Fig. 10). These levels of increased

blood pressure are similar to those reported in transgenic miceglobally expressing ECFP/ANG II (29) or in AT1a-KO micewith the knockin of AT1a receptors selectively in proximaltubules (13), but much lower than those induced by chronicinfusion 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 ofthe kidney increases blood pressure remain to be determined.In cultured COS-7 or CHO-K1 cells, coexpression of ECFP/ANG II with the AT1 receptor fused to enhanced yellowfluorescent protein, AT1R/EYFP, significantly induced cellproliferation via activation of cAMP response element-associ-ated protein (CREB) activity (4) or in A10 vascular smoothmuscle cells, ECFP/ANG II activated p38 MAP kinase (3).Both effects were inhibited by losartan, suggesting an AT1

receptor-dependent response. Baker et al. (1, 2, 12) demon-strated that expression of an intracellular ANG II peptide incardiac myocytes in vitro or in vivo induced a hypertrophiceffect, a response that does not require activation of AT1

receptors. Our present study strongly suggests that the bloodpressure-increasing effect of ECFP/ANG II expression selec-tively in proximal tubules of the kidney is specific and depen-dent on AT1 (AT1a) receptor activation. Indeed, intrarenaladenoviral transfer of a control scrambled sequence of ECFP/ANG IIc selectively in proximal tubules using the same sglt2promoter did not alter blood pressure in rats (Fig. 10). Further-more, concurrent treatment of the rats with losartan completelynormalized systolic blood pressure elevated by proximal tu-bule-specific transfer of ECFP/ANG II. Finally, the bloodpressure effect of ECFP/ANG II transfer was completely abol-ished in AT1a receptor-KO mice (Fig. 10).

Although how ECFP/ANG II increases blood pressurethrough activation of intracellular AT1 (AT1a) receptors inproximal tubules is not known, the present study suggests thatthe effect may be in part mediated by increasing fluid andsodium reabsorption in proximal tubules via AT1a-mediatedtranscriptional effect on NHE-3 expression. We have recentlyshown that ANG II directly stimulated AT1a receptors toinduce transcriptional responses of NHE-3 in freshly isolatedrat renal cortical nuclei (19) and cultured proximal tubule cells(14, 17). Furthermore, expression of ECFP/ANG II in culturedmouse proximal tubule cells increases NHE-3 protein expres-sion in wild-type, but not AT1a-KO, mouse proximal tubulecells (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 ofthe kidney on body and kidney weights, 24-h drinking, and urinary excretion of water and electrolytes in rats 14 days afterECFP/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 � 8Left kidney wt 1.42 � 0.06 1.41 � 0.06 1.46 � 0.08 1.40 � 0.04Left kidney wt-to-body wt ratio �100 0.38 � 0.05 0.38 � 0.07 0.41 � 0.08 0.36 � 0.04Drinking, ml/24 h 40.1 � 1.6 43.6 � 2.3 42.4 � 1.64 40.5 � 1.2V, ml/24 h 20.0 � 0.42 16.30 � 0.36* 21.84 � 0.55† 18.2 � 0.3UNaV, mmol/24 h 2.14 � 0.04 1.76 � 0.04* 2.33 � 0.06† 2.10 � 0.02UKV, 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 significancesin 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-specific transfer of ECFP/ANG II or itsscrambled control, ECFP/ANG IIc, with or without losartan treatment onsystolic blood pressure in rats (SBP; A) or ECFP/ANG II transfer in wild-typeor AT1a-KO mice (B). *P � 0.05 or **P � 0.01 vs. basal SBP. P � 0.05 orP � 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 IItransfer.




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since ANG II appears to increase ROS in freshly isolated ratand sheep renal cortical nuclei, which is also AT1 receptordependent and may have long-term genomic effects. IncreasedNHE-3 expression or activity in proximal tubules by intracel-lular ANG II may lead to increases in proximal tubule sodiumtransport and decreases in 24-h urinary sodium excretion. In thepresent study, we carefully housed the animals individually in ametabolic cage and provided the same ration of food and waterto maintain the same level of food and water intake for all rats.Under these conditions, fractional sodium and lithium excre-

tion was significantly 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 ofwhole 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-specific trans-fer of ECFP/ANG II in rats or mice increases blood pressureprimarily via activation of AT1 (AT1a) receptors to increaseproximal tubule sodium reabsorption in the kidney.

However, the present study may have some limitations. Forexample, unlike the transgenic mouse model (29), our ap-proach is adenovirus mediated, and the effects of ECFP/ANGII transfer on blood pressure and proximal tubular functionunlikely last a lifetime. Second, we may not completely ex-clude the possibility that ECFP/ANG II may interact with cellsurface receptors at the intracellular side. If this occurred,ECFP/ANG II may activate cell surface receptors in a mannersimilar to extracellular ANG II. In live cell fluorescenceimaging studies, we did not visualize ECFP/ANG II secretioninto the medium or trafficked to the cell membranes. Third, toincrease the efficiency or effectiveness of the gene transferselectively in proximal tubules of the kidney, blood flow to thekidney would preferably be disrupted temporarily for severalminutes. This may lead to ischemic renal injury and thereforeelevate blood pressure. This seems unlikely since we con-firmed that temporary disruption of renal blood flow for 5 mindid not cause apparent pathological changes in the kidneys ofcontrol or ECFP/ANG II-transferred rats in preliminary stud-ies. Other investigators have reported no irreversible kidneyinjury with this gene transfer procedure (22, 23). Finally,although at its peak intrarenal adenoviral transfer of this ANGII fusion protein selectively in proximal tubules decreasedfractional sodium and lithium excretion while increasing bloodpressure, it may be difficult to infer that increased bloodpressure was directly due to sodium retention. Further studiesto simultaneously monitoring daily sodium balance and bloodpressure homeostasis in response to ECFP/ANG II transfer orto directly study sodium transport responses in isolated prox-imal tubule preparations may be necessary to determine thecause and blood pressure effect relationship.

In summary, the present study demonstrates for the first timethat recombinant human adenovirus and the sglt2 gene pro-moter can effectively drive the expression of an intracellularcyan fluorescent fusion of ANG II protein selectively in prox-

Fig. 11. Effect of proximal tubule-specific transfer of ECFP/ANG II in thekidney on fractional sodium (FENa) and lithium excretion (FELi) in ECFP/ANG II-transferred rats 2 wk after the gene transfer. Fractional lithiumexcretion was used as an indirect index of proximal tubular sodium reabsorp-tion in the entire kidney (36, 37). A decrease in fractional lithium excretionsuggests an increase in overall proximal tubular sodium reabsorption in thekidney. **P � 0.01 vs. the control group without ECFP/ANG II transfer at day14. P � 0.01 vs. ECFP/ANG II-transferred rats.

Table 4. Long-term effects of proximal tubule-specific transfer of ECFP/ANG II in the rat kidney on body and left kidneyweights, systolic blood pressure, and 24-h urinary excretion of water and sodium 28 days after intrarenal ECFP/ANG IItransfer

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 � 0.0Heart wt, g 1.39 � 0.04 1.43 � 0.04SBP, 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.




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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. Noapparent ectopic ECFP/ANG II expression was observed inextrarenal tissues, thereby confirming the proximal tubulecell-specific nature of the approach. The expression of ECFP/ANG II in proximal tubules of one kidney appears to besufficient to elevate blood pressure, and the effect is likelymediated by activation of AT1 (AT1a) receptors and increasesin sodium and fluid reabsorption in proximal tubules. Furtherstudies using isolated proximal tubule preparations or an invivo micropuncture technique in rats or mice with proximaltubule-specific transfer of this intracellular ANG II fusionprotein may be necessary to further elucidate the physiologicaleffects and underlying cellular mechanisms of intracellularANG II on blood pressure regulation.

ACKNOWLEDGMENTS

Portions of this work were presented at the 63rd High Blood PressureResearch Council Conference of the American Heart Association in Chicago,IL, September 23–26, 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 AmericanSociety of Nephrology M. James Scherbenske Grant, and institutional supportfrom the Henry Ford Health System and the University of Mississippi MedicalCenter to J. L. Zhuo. J. L. Cook was supported by NIH Grant R01 HL072795.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

REFERENCES

1. Baker KM, Chernin MI, Schreiber T, Sanghi S, Haiderzaidi S, BoozGW, Dostal DE, Kumar R. Evidence of a novel intracrine mechanism inangiotensin II-induced cardiac hypertrophy. Regul Pept 120: 5–13, 2004.

2. Baker KM, Kumar R. Intracellular angiotensin II induces cell prolifer-ation independent of AT1 receptor. Am J Physiol Cell Physiol 291:C995–C1001, 2006.

3. Cook JL, Mills SJ, Naquin R, Alam J, Re RN. Nuclear accumulation ofthe AT1 receptor in a rat vascular smooth muscle cell line: effects uponsignal transduction and cellular proliferation. J Mol Cell Cardiol 40:696–707, 2006.

4. Cook JL, Re R, Alam J, Hart M, Zhang Z. Intracellular angiotensin IIfusion protein alters AT1 receptor fusion protein distribution and activatesCREB. J Mol Cell Cardiol 36: 75–90, 2004.

5. Cook JL, Zhang Z, Re RN. In vitro evidence for an intracellular site ofangiotensin action. Circ Res 89:1138–1146, 2001.

6. de Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T. InternationalUnion of Pharmacology. XXIII. The angiotensin II receptors. PharmacolRev 52: 415–472, 2000.

7. Ding Y, Davisson RL, Hardy DO, Zhu LJ, Merrill DC, Catterall JF,Sigmund CD. The kidney androgen-regulated protein promoter confersrenal proximal tubule cell-specific and highly androgen-responsive expres-sion on the human angiotensinogen gene in transgenic mice. J Biol Chem272: 28142–28148, 1997.

8. Gwathmey T, Shaltout HA, Pendergrass KD, Pirro NT, Figueroa JP,Rose JC, Diz DI, Chappell MC. Nuclear angiotensin II type 2 (AT2)receptors are functionally linked to nitric oxide production. Am J PhysiolRenal Physiol 296: F1484–F1493, 2009.

9. Gwathmey TM, Pendergrass KD, Reid SD, Rose JC, Diz DI, ChappellMC. Angiotensin-(1–7)-angiotensin-converting enzyme 2 attenuates reac-tive oxygen species formation to angiotensin II within the cell nucleus.Hypertension 55:166–71, 2010.

10. Haller H, Lindschau C, Erdmann B, Quass P, Luft FC. Effects ofintracellular angiotensin II in vascular smooth muscle cells. Circ Res 79:765–772, 1996.

11. Hill JV, Findon G, Appelhoff RJ, Endre ZH. Renal autoregulation andpassive pressure-flow relationships in diabetes and hypertension. Am JPhysiol Renal Physiol 299: F837–F844, 2010.

12. Kumar R, Singh VP, Baker KM. The intracellular renin-angiotensinsystem: a new paradigm. Trends Endocrinol Metab 18: 208–214, 2007.

13. Le TH, Oliverio MI, Kim HS, Salzler H, Dash RC, Howell DN,Smithies O, Bronson S, Coffman TM. A �GT-AT1A receptor transgeneprotects renal cortical structure in AT1 receptor-deficient mice. PhysiolGenomics 18: 290–298, 2004.

14. Li XC, Hopfer U, Zhuo JL. AT1 receptor-mediated uptake of angiotensinII and NHE-3 expression in proximal tubule cells through the microtubule-dependent endocytic pathway. Am J Physiol Renal Physiol 297: F1342–F1352, 2009.

15. Li XC, Navar LG, Shao Y, Zhuo JL. Genetic deletion of AT1a receptorsattenuates intracellular accumulation of angiotensin II in the kidney ofAT1a receptor-deficient mice. Am J Physiol Renal Physiol 293: F586–F593, 2007.

16. Li XC, Shao Y, Zhuo JL. AT1a receptor knockout in mice impairs urineconcentration by reducing basal vasopressin levels and its receptor sig-naling proteins in the inner medulla. Kidney Int 76: 169–177, 2009.

17. Li XC, Zhuo JL. Selective knockdown of AT1 receptors by RNAinterference inhibits Val5-ANG II endocytosis and NHE-3 expression inimmortalized rabbit proximal tubule cells. Am J Physiol Cell Physiol 293:C367–C378, 2007.

18. Li XC, Zhuo JL. In vivo regulation of AT1a receptor-mediated intracel-lular uptake of [125I]-Val5-angiotensin II in the kidneys and adrenal glandsof AT1a receptor-deficient mice. Am J Physiol Renal Physiol 294: F293–F302, 2008.

19. Li XC, Zhuo JL. Intracellular ANG II directly induces in vitro transcrip-tion of TGF-�1, MCP-1, and NHE-3 mRNAs in isolated rat renal corticalnuclei via activation of nuclear AT1a receptors. Am J Physiol Cell Physiol294: C1034–C1045, 2008.

20. Mattson DL, Kunert MP, Roman RJ, Jacob HJ, Cowley AW Jr.Substitution of chromosome 1 ameliorates L-NAME hypertension andrenal disease in the fawn-hooded hypertensive rat. Am J Physiol RenalPhysiol 288: F1015–F1022, 2005.

21. Mehta PK, Griendling KK. Angiotensin II cell signaling: physiologicaland pathological effects in the cardiovascular system. Am J Physiol CellPhysiol 292: C82–C97, 2007.

22. Moullier P, Friedlander G, Calise D, Ronco P, Perricaudet M, FerryN. Adenoviral-mediated gene transfer to renal tubular cells in vivo. KidneyInt 45: 1220–1225, 1994.

23. Ortiz PA, Hong N, Plato CF, Varela M, Garvin JL. An in vivo methodfor adenovirus-mediated transduction of thick ascending limbs. Kidney Int63: 1141–1149, 2003.

24. Ortiz RM, Graciano ML, Seth D, Awayda MS, Navar LG. Aldosteronereceptor antagonism exacerbates intrarenal angiotensin II augmentation inANG II-dependent hypertension. Am J Physiol Renal Physiol 293: F139–F147, 2007.

25. Pendergrass KD, Averill DB, Ferrario CM, Diz DI, Chappell MC.Differential expression of nuclear AT1 receptors and angiotensin II withinthe kidney of the male congenic mRen2 Lewis rat. Am J Physiol RenalPhysiol 290: F1497–F1506, 2006.

26. Pendergrass KD, Gwathmey TM, Michalek RD, Grayson JM, Chap-pell MC. The angiotensin II-AT1 receptor stimulates reactive oxygenspecies within the cell nucleus. Biochem Biophys Res Commun 384:149–154, 2009.

27. Re RN. On the biological actions of intracellular angiotensin. Hyperten-sion 35: 1189–1190, 2000.

28. Redding KM, Chen BL, Singh A, Re RN, Navar LG, Seth DM,Sigmund CD, Tang WW, Cook JL. Transgenic mice expressing anintracellular fluorescent fusion of angiotensin II demonstrate renal throm-botic microangiopathy and elevated blood pressure. Am J Physiol HeartCirc Physiol 298: H1807–H1818, 2010.

29. Rubera I, Poujeol C, Bertin G, Hasseine L, Counillon L, Poujeol P,Tauc M. Specific Cre/Lox recombination in the mouse proximal tubule. JAm Soc Nephrol 15: 2050–2056, 2004.

30. Timmermans PB, Wong PC, Chiu AT, Herblin WF, Benfield P, CariniDJ, Lee RJ, Wexler RR, Saye JA, Smith RD. Angiotensin II receptorsand angiotensin II receptor antagonists. Pharmacol Rev 45: 205–251,1993.




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31. van Esch JH, Gembardt F, Sterner-Kock A, Heringer-Walther S, LeTH, Lassner D, Stijnen T, Coffman TM, Schultheiss HP, Danser AH,Walther T. Cardiac phenotype and angiotensin II levels in AT1a, AT1b,and AT2 receptor single, double, and triple knockouts. Cardiovasc Res 86:401–409, 2010.

32. van Kats JP, Methot D, Paradis P, Silversides DW, Reudelhuber TL.Use of a biological peptide pump to study chronic peptide hormone actionin transgenic mice. Direct and indirect effects of angiotensin II on theheart. J Biol Chem 276: 44012–44017, 2001.

33. Vinay P, Gougoux A, Lemieux G. Isolation of a pure suspension of ratproximal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 241:F403–F411, 1981.

34. Xu J, Carretero OA, Lin CX, Cavasin MA, Shesely EG, Yang JJ,Reudelhuber TL, Yang XP. Role of cardiac overexpression of ANG II inthe regulation of cardiac function and remodeling postmyocardial infarc-tion. Am J Physiol Heart Circ Physiol 293: H1900–H1907, 2007.

35. Zhuo JL, Allen AM, Alcorn D, MacGregor D, Aldred GP, Mendel-sohn FA. The distribution of angiotensin II receptors. In: Hypertension:

Pathology, Diagnosis & Management, edited by Laragh JH and BrennerBM (2nd ed.). New York: Raven, 1739–1762, 1995.

36. Zhuo JL, Thomas D, Harris PJ, Skinner SL. The role of endogenousangiotensin II in the regulation of renal haemodynamics and proximal fluidreabsorption in the rat. J Physiol 453: 1–13, 1992.

37. Zhuo JL, Harris PJ, Skinner SL. Atrial natriuretic factor modulatesproximal glomerulotubular balance in anesthetized rats. Hypertension 14:666–673, 1989.

38. Zhuo JL, Imig JD, Hammond TG, Orengo S, Benes E, Navar LG.ANG II accumulation in rat renal endosomes during ANG II-inducedhypertension: role of AT1 receptor. Hypertension 39: 116–121, 2002.

39. Zhuo JL, Li XC. Novel roles of intracrine angiotensin II and signallingmechanisms in kidney cells. J Renin Angiotensin Aldosterone Syst 8:23–33, 2007.

40. Zhuo JL, Li XC, Garvin JL, Navar LG, Carretero OA. Intracellularangiotensin II induces cytosolic Ca2 mobilization by stimulating intra-cellular AT1 receptors in proximal tubule cells. Am J Physiol RenalPhysiol 290: F1382–F1390, 2006.




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