Kidney International, Vol. 63 (2003), pp. 1141–1149

An in vivo method for adenovirus-mediated transduction ofthick ascending limbs

PABLO A. ORTIZ, NANCY J. HONG, CRAIG F. PLATO, MARISELA VARELA,and JEFFREY L. GARVIN

Division of Hypertension and Vascular Research, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan

An in vivo method for adenovirus-mediated transduction of The thick ascending limb of the loop of Henle (THAL)thick ascending limbs. reabsorbs 30% of filtered sodium chloride (NaCl) along

Background. The thick ascending limb of the loop of Henle with most of the bicarbonate that escapes proximal tu-(THAL) plays an important role in the maintenance of salt, bule reabsorption [1, 2]. Due to the important role playedwater, and acid-base balance. While techniques for gene trans-

by the THAL in salt, water, and acid-base homeostasis,fer of renal vascular cells and some tubular segments haveion transport in this segment is highly regulated. Genebeen described, in vivo transduction of THALs has not beenmutations of THAL transporters that result in decreasedsuccessful. We hypothesized that in vivo injection of adenoviralNaCl reabsorption cause severe defects in urinary con-vectors into the renal medulla would result in efficient transduc-

tion of THALs. centrating ability in humans [3–5]. In addition, defectiveMethods. We injected recombinant adenoviruses containing regulation of NaCl absorption by THALs has been asso-

the reporter gene, green fluorescent protein (GFP), driven by ciated with the development of salt-sensitive hyperten-either the cytomegalovirus promoter (Ad-CMVGFP) or the sion in rats [6–8].promoter for the Na/K/2 Cl cotransporter (Ad-NKCC2GFP), Different experimental approaches are currently beingwhich is THAL-specific, into the outer medullary interstitium

used to study the regulation and mechanisms of ion trans-of Sprague-Dawley rat kidneys. Kidneys were removed at vari-port in the THAL. In the last decade, methods for geneticous times after viral injection and analyzed for GFP expression.manipulation of animals have been developed for use asResults. Western blots revealed strong GFP expression in

the outer medulla (which is composed primarily of THALs) 5 research tools. Gene knockout technology has proven todays after Ad-CMVGFP injection. We quantified THAL trans- be an excellent research tool and has been used to studyduction efficiency by scoring the number of fluorescent tubules regulation of transport in the THAL and other nephronin THALs suspensions, which showed that at least 77 � 3% segments [9–11]. However, gene knockout technologyof THAL expressed GFP. To specifically transduce THALs, we has primarily been successful in mice, with very limitedinjected Ad-NKCC2GFP into the medullary interstitium. As

success in rats. Other methods for genetic manipulationdetermined by Western blot, GFP expression was only detectedextensively used in animals involve somatic transfer ofin the outer medulla. Immunohistochemistry and confocal mi-DNA or RNA using vectors such as liposomes, adeno-croscopy showed that GFP was localized to tubular cells posi-viruses, and adeno-associated viruses and retroviruses.tive for Tamm-Horsfall protein. Thus, GFP fluorescence was

only detected in THALs, not in cortical, inner medulla or vascu- Techniques for in vivo gene transfer to the kidney havelar cells. Time-course studies showed that GFP expression in led to successful transduction of glomeruli, endothelialTHALs was measurable from 4 to 14 days, peaked at 7 days, cells, proximal tubules [12–15], and inner medullary col-and had returned to background levels by 21 days. lecting ducts [15, 16]. However, to our knowledge, in

Conclusion. This method facilitates highly efficient, THAL- vivo transduction of the THAL has not been reported.specific transduction. While application of this technique forWe have designed a versatile and highly efficient tech-gene therapy in humans is unlikely due to the transient genenique for adenovirus-mediated in vivo transduction ofexpression observed and the impossibility for repeated injec-THAL cells that can be used for a wide range of studies.tions of adenoviral vectors, this method provides a valuable

tool for investigators studying regulation and mechanisms of This technique should be very useful to study the roleTHAL ion transport and its relationship to whole-kidney physi- of a specific protein in regulating THAL function in vitroology and pathophysiology. and in vivo, something previously possible only in cell

culture systems.

Key words: kidney tubules, hypertension, NaCl transport.METHODS

Received for publication April 25, 2002 Adenoviral vectorsand in revised form August 23, 2002, and October 3, 2002Accepted for publication October 22, 2002 Recombinant replication-deficient adenoviruses con-

taining the reporter gene green fluorescent protein (GFP) 2003 by the International Society of Nephrology

1141

Ortiz et al: In vivo transduction of THAL1142

Fig. 2. Transduction efficiency of medullary thick ascending limbs(mTHALs) from rat kidneys injected with the reporter gene greenfluorescent protein (GFP) under the control of the cytomegalovirus(CMV) promoter (Ad-CMVGFP). GFP was expressed in 77 � 3%of mTHALs as observed by fluorescence microscopy. Kidneys wereremoved 7 days after injection (N � 4).

under the control of either the cytomegalovirus (CMV)promoter (Ad-CMVGFP) or the Na/K/2 Cl (NKCC2) co-transporter promoter (Ad-NKCC2GFP) were constructedby the University of Iowa Gene Transfer Vector Coreas described previously [17, 18]. The cDNA for GFP wascloned into a shuttle vector pAdCMVKpn-Not1, whichcontains a CMV promoter, a cloning site for insertionof a heterologous gene, and a polyadenylation signalflanked by adenoviral sequences 5� and 3�. The 5� endcontains the first 360 bp of the left hand of the adenoviralgenome, including the packaging signal. The 3� end con-tains approximately 2.5 kb of flanking adenoviral DNAto facilitate homologous recombination. The same plas-mid vector was used to generate Ad-NKCC2GFP, exceptthat the CMV promoter was replaced by a 1.8 kb segmentof the 5� flanking region of the Nkcc2 gene [19]. Theadenoviral shuttle plasmids were transfected into thepermissive HEK 293 host cell line along with nearly full-length adenoviral DNA that was restricted to removethe E1 region. Virus isolates were plaque-purified andpropagated in HEK 293 cells, isolated, concentrated, andtitered.

In vivo kidney transductionFig. 1. (A) Representative composites of four low-power (�40) micro- Male Sprague-Dawley rats weighing 120 to 150 ggraphs from the left kidney of a rat 5 days after injections of the (Charles River Breeding Laboratories, Wilmington, MA,reporter gene green fluorescent protein (GFP) under the control of

USA) were fed a diet containing 0.22% sodium and 1.1%the cytomegalovirus (CMV) promoter (Ad-CMVGFP) (N � 4). (B)Representative Western blot of homogenates from the cortex and outer potassium (Purina, Richmond, IN, USA). On the day ofmedulla of a rat kidney transduced with Ad-CMVGFP. Samples were surgery, rats were weighed to determine how deep toprepared 5 days postinjection. Eighty micrograms of protein were

insert the needle, as the tip has to be positioned betweenloaded per lane. Exposure time was 5 minutes. Molecular weight mark-ers are shown on the left (N � 3). the corticomedullary junction and the inner stripe of the

Ortiz et al: In vivo transduction of THAL 1143

50 tubing connected to a nanoliter syringe pump (Har-vard Apparatus, Holliston, MA, USA) set at 20 �L/min. The needle was inserted perpendicular to the renalcapsule and parallel to the medullary rays and aimedtoward the medulla. Injections were made along a straightline within the capsule, with two injection points sepa-rated by approximately 2.5 mm. To avoid leakage of thevirus, the needle was not removed until 30 seconds aftereach injection. The tubing was prefilled with mineral oilto ensure complete delivery of the virus. The clamp wasremoved from the renal artery and vein after 8 minutes.Once renal blood flow was reestablished, the kidney wasreturned to the abdominal cavity and the incision wasclosed. Survival was greater than 90%.

Western blot of GFP expression

Kidneys transduced with Ad-CMVGFP were removed5 days after injection, while those transduced with Ad-NKCC2GFP were removed at 4, 7, 14, or 21 days post-injection for time-course analysis. The lung, liver, andspleen of rats injected with Ad-NKCC2GFP were har-vested 7 days after the injections. Sections of the outermedulla and either the cortex or inner medulla wereexcised from coronal slices, minced, and homogenizedin 0.5 mL of a buffer containing 20 mmol/L N-2-hydroxy-ethylpiperazine-N�-2-ethanesulfonic acid (HEPES) (pH7.4), 2 mmol/L ethylenediaminetetraacetic acid (EDTA),0.3 mol/L sucrose, and a protease inhibitor cocktail, using

Fig. 3. (A) Representative Western blot of homogenates from the cor- a glass tissue grinder (six strokes). Cell debris was re-tex and outer medulla of a rat kidney transduced with the Na/K/2 Cl moved by centrifugation, and an aliquot of the superna-(NKCC2) cotransporter promoter (Ad-NKCC2GFP) (N � 4). Samples

tant was used to determine protein concentration usingwere prepared 7 days postinjection. Protein (80 �g) was loaded perlane. Samples were exposed overnight. Molecular weights of marker Coomassie protein assay reagent (Pierce, Rockford, IL,proteins are shown on the left. Abbreviations are: C1, cortex, rat #1; USA). The same procedure was used to obtain cell ho-M1, outer medulla, rat #1; C2, cortex, rat #2; M2, outer medulla, rat

mogenates from other tissues. Protein (80 �g) was sepa-#2. (B) Representative Western blot of homogenates from the lung(LU), liver (LI), and spleen (SP) of a rat were given renal injections of rated out by electrophoresis on a 12% polyacrylamideAd-NKCC2GFP (N � 2). Samples were prepared 7 days postinjection. gel and electrotransferred to a polyvinylidine difluorideEighty micrograms of protein were loaded per lane. Samples were

(PVDF) membrane (Millipore, Bedford, MA, USA).exposed overnight. Molecular weight markers are shown on the left.Lane 1 is a positive green fluorescent protein (GFP) control. Nonspecific binding was blocked by incubating the mem-

brane for 1 hour with 5% nonfat dry milk in Tris-bufferedsaline-Tween (TBS-T). TBS-T contained 50 mmol/L Tris(pH 7.5), 500 mmol/L NaCl, and 0.1% Tween 20. The

outer medulla. For this purpose, the distance from the membrane was then incubated for 1 hour with a 1:200renal capsule to the corticomedullary junction was mea- dilution of GFP antibody isolated from mice (Santa Cruzsured in coronal sections of 30 rats weighing 100 to 200 g, Biotechnology, Santa Cruz, CA, USA), and then washedplotted against body weight and subjected to regression with TBS-T. After 1-hour incubation with a 1:1000 dilu-analysis. A plastic sleeve was glued onto the needle at tion of antimouse immunoglobulin (Ig)-horseradish per-a point corresponding to the desired depth. Animals were oxidase (HRP)-linked antibody (Amersham, Arlingtonanesthetized with ketamine (80 mg/kg body weight intra- Heights, IL, USA), the membrane was washed againperitoneally) and xylazine (10 mg/kg body weight intra- and GFP detected using enhanced chemiluminescenceperitoneally), shaved, and cleaned with povidone-iodine (ECL) Western reagents (Amersham, Piscataway, NJ,(Betadine). The left kidney was exposed via a flank inci- USA). All incubations were performed at room tempera-sion, the fatty tissue removed, and both the renal artery ture. Densitometry of GFP bands was performed usingand vein were clamped. Immediately, four 20 �L undi- Molecular Analyst software (Biorad Laboratories, Her-luted virus injections (1 to 1.2 � 1012 particles/mL) were cules, CA, USA), and the results were expressed as arbi-

trary units per milligram of protein.made using a 30-gauge needle attached to polyethylene-

Fig. 5. (A) Confocal fluorescence micrographs showing specific greenfluorescent protein (GFP) expression in medullary thick ascending limbs(THAL) from a rat kidney transduced with the Na/K/2 Cl (NKCC2)cotransporter promoter (Ad-NKCC2GFP) (scale bar � 25 �m). (Band C ) Fluorescent and transmittance micrographs showing specificGFP expression in a cortical THAL from a kidney transduced withAd-NKCC2GFP (N � 4). Green , GFP; red, Tamm-Horsfall (scalebar � 25 �m).

Ortiz et al: In vivo transduction of THAL 1145

Fig. 4. Confocal fluorescence (A) and transmittance (B) micrographs showing specific GFP expression in medullary thick ascending limbs (THAL)from a rat kidney transduced with the Na/K/2 Cl (NKCC2) cotransporter promoter (Ad-NKCC2GFP). Green, green fluorescent protein (GFP);red, Tamm-Horsfall. No green fluorescence was observed in the cortex and inner medulla. Magnification � 400 (scale bar � 20 �m).�

Transduction efficiency in THAL suspensions temperature with a 1:100 dilution of an antibody againstgoat antihuman Tamm-Horsfall protein (ICN Cappel,Suspensions of medullary THALs (mTHALs) wereAurora, OH, USA), and then for 1 hour at room temper-prepared as described previously [20]. Kidneys were per-ature with a 1:400 dilution of secondary antibody (Alexafused retrograde via the aorta with a solution containingFluor 568 donkey antigoat IgG, Molecular Probes, Eu-0.1% collagenase and 100 U heparin. The inner stripegene, OR, USA). GFP fluorescence was detected withof the outer medulla was cut from coronal slices, minced,a confocal laser scanning system (Noran Instruments,and incubated at 37�C for 30 minutes in 0.1% collagen-Middletown, WI, USA) set at 488 nm excitation with aase. The tissue was pelleted via low-speed centrifugation,500 nm long-pass filter (exposure time � 35 seconds).resuspended in cold perfusion solution, filtered throughThe same settings were used to detect Tamm-Horsfalla 250 �m nylon mesh, and centrifuged. The pellet was

washed, centrifuged again, and finally resuspended in protein, except that 568 nm excitation and a 605/55 band0.2 mL cold perfusion solution. Samples of mTHAL sus- pass (BP) filter were used. Images from the cortex, outerpensions were placed on a microscope slide and viewed medulla, and inner medulla were colored using a fluo-with a fluorescence microscope equipped with a xenon rescein isothiocyanate (FITC) pseudocoloring option forarc lamp, a 470 nm filter (20 nm bandwidth), an image GFP and rhodamine pseudocoloring for Tamm-Horsfallintensifier (Video Scope International, Herndon, VA, protein. Transduction efficiency was quantified in trans-USA) and a charge-coupled device (CCD) camera (Ha- verse (cross-section) slices of the renal medulla. A totalmamatsu, Hamamatsu City, Japan). Light microscopy of 1000 THALs in 12 random, nonoverlapping fieldswas used to count the total number of mTHALs in the (�20) were counted per slide. THALs positive forsample. Those tubules that exhibited fluorescence when Tamm-Horsfall protein and negative for GFP were con-excited at 470 nm were scored as expressing GFP. Most sidered “not transduced.” The confidence interval fortubules (�90%) were either entirely negative or entirely N � 1000 was less than �2%.positive. Fewer than 10% of the tubules contained both Low-power micrographs were obtained from coronalpositive and negative cells. These tubules were scored slices of kidneys injected with Ad-CMVGFP. Briefly, 5as positive if at least 75% of cells along the tubule were days after viral injection, rats were anesthetized and thefluorescent and were scored negative if fewer than 75% left kidney flushed via the renal artery with cold perfu-of cells were fluorescent. A total of 50 tubules were

sion solution containing a protease inhibitor “cocktail.”counted per suspension. Transduction efficiency was cal-

The solution composition was (in mmol/L) 114 NaCl,culated as the percentage of tubules that exhibited fluo-

4.0 KCl, 25 NaHCO3, 2.5 NaH2PO4, 1.2 MgSO4, 1.0 Carescence. The confidence interval was less than �12%lactate, 5.5 glucose, 6.0 alanine, and 1.0 Na3 citrate, equili-when 50 tubules were sampled.brated with 95% O2-5% CO2. Osmolality was adjusted to290 � 3 mOsm/kg H2O. The protease inhibitor “cocktail”Confocal microscopy and histologic analysisincluded (in �g/mL) 5 antipain, 10 aprotinin, 5 leupeptin,Seven days after viral injection, kidneys transduced5 chymostatin, 2.5 pepstatin A, and 25 4-(2-aminoethyl)with Ad-NKCC2GFP were preserved by retrograde per-benzenesulfonyl fluoride hydrochloride (Sigma Chemi-fusion of the aorta with 150 mmol/L NaCl to flush thecal Company, St. Louis, MO, USA). The kidney waskidney, followed by a 15-minute perfusion with 4% para-removed, and coronal slices 1 to 2 mm thick were viewedformaldehyde in buffer containing 150 mmol/L NaCl andwith a Nikon fluorescence microscope equipped with a10 mmol/L sodium phosphate (pH 7.4). Kidneys were470 nm excitation filter (20 nm bandwidth). Images werestored in 4% formaldehyde until sectioning, at whichsaved using a SPOT camera and imaging software (Diag-point they were embedded in paraffin and cut into slicesnostic Instruments, Sterling Heights, MI, USA).5 �m thick. Longitudinal and transverse sections of the

cortex and medulla were obtained from different kid-Statisticsneys. To expose antigenic sites, fixed paraffin–embedded

Results are expressed as mean � SEM. ANOVAslices were first deparaffinized with xylene and then hy-(analysis of variance) was used to determine statisticaldrated gradually through 100% ethanol, then 95%, 70%,differences in GFP expression. P � 0.05 was consideredand finally distilled water. Each lasted for 5 minutes.

Slides were air-dried and incubated for 2 hours at room significant.

Ortiz et al: In vivo transduction of THAL1146

To determine transduction efficiency in THALs, weprepared suspensions of mTHALs from kidneys injectedwith Ad-CMVGFP. We then used fluorescence micros-copy to count the number of THALs that exhibited fluo-rescence out of a total number of 50 tubules per sample.Fluorescence was observed along the tubule and in mostcells of the transduced THALs. We found that 77 � 3% ofTHALs expressed GFP (N � 4) (Fig. 2). The confidenceinterval was less than �12%. These data indicate thatdirect injection of viral vectors into the medullary inter-stitium results in efficient transduction of THALs.

Since our objective was to selectively express a trans-gene in THALs, we tested our method using a cell type-specific promoter to drive GFP expression only in THALs.For this purpose, we used the promoter for the genecoding the bumetanide-sensitive Na/K/2Cl transporter(NKCC2), which is only expressed in the THAL [19, 21,22]. To test whether transduction was restricted to theTHAL and show that the NKCC2 promoter was specific,kidney sections from injected rats were analyzed byWestern blot and confocal microscopy. Western blotsshowed GFP expression only in the outer medulla, notin the cortex (N � 4) (Fig. 3A). In addition, GFP expres-sion was not detected in the liver, lung, or spleen of rats7 days after injection of Ad-NKCC2GFP into the kidney(Fig. 3B).

Because Western blots did not indicate which cells inthe outer medulla expressed GFP, we next analyzed trans-duced kidneys with immunohistochemistry and confocalmicroscopy. THALs were identified by labeling Tamm-Horsfall protein [23]. In longitudinal sections along the

Fig. 6. (A) Time course of green fluorescent protein (GFP) expression tubular axis, GFP fluorescence (green) was only ob-in outer medullary homogenates from rat kidneys transduced with The served in tubules showing Tamm-Horsfall protein (red)Na/K/2 Cl (NKCC2) cotransporter promoter (Ad-NKCC2GFP). Re-

(Fig. 4). No GFP fluorescence was detected in any corti-sults are expressed as mean � SEM. Numbers of rats used for eachtime point were 4 days (N � 4); 7 days (N � 6); 14 days (N � 4); and cal or inner medullary structures when medullary injec-21 days (N � 4). (B) Representative Western blot of homogenates tions were performed (data not shown).from the cortex and outer medulla of a kidney transduced with Ad-

Transduction efficiency was also determined in theNKCC2GFP 21 days postinjection (N � 4). Eighty micrograms of pro-tein were loaded per lane. Samples were exposed overnight. Molecular medulla of kidneys injected with Ad-NKCC2GFP. Trans-weight markers are shown on the left. 1, cortex, rat #1; 2, outer medulla, verse (cross-section) slices were immunolabeled for Tamm-rat #1; 3, cortex, rat #2; 4, outer medulla, rat #2; 5, outer medulla,

Horsfall protein and analyzed by confocal microscopy.noninjected rat kidney (negative control).We counted 1000 THALs positive for Tamm-Horsfall,and those also showing green fluorescence were scored astransduced. We found that 91 � 3% of THALs expressedGFP (Fig. 5). The confidence interval was less than �2%.RESULTSTo further test the specificity of the NKCC2 promoter,In order to maximize exposure of THAL cells to thewe injected Ad-NKCC2GFP into the renal cortex. Sevenadenoviral vectors, we injected the vectors directly intodays after injection, transverse (cross-section) slices ofthe interstitium of the outer medulla. Five days afterthe cortex were analyzed for Tamm-Horsfall and GFPinjecting Ad-CMVGFP, GFP expression was found inexpression. A small percentage of cortical THAL cellsall cells around the injection site as observed by fluores-that were positive for Tamm-Horsfall also exhibitedcence microscopy (N � 4) (Fig. 1A). When GFP expres-green fluorescence. No GFP was detected in proximalsion was analyzed in kidney slices by Western blot (Fig.or distal tubules or in either vascular or glomerular cells1B), a strong band was detected for the outer medulla,(N � 4) (Fig. 5B). Histologic analysis revealed no mor-and a weaker band was found in the cortex. These resultsphologic abnormalities in the kidneys injected with Ad-confirmed that transduction occurred primarily in the

area where the tip of the needle was positioned (N � 3). NKCC2GFP.

Ortiz et al: In vivo transduction of THAL 1147

Finally, we used Western blot to study the time course when 50 tubules were counted, and it overlapped with theconfidence interval of �2% obtained when 1000 THALsof GFP expression in the outer medulla of kidneys trans-

fected with Ad-NKCC2GFP (Fig. 6A). At 4 days post- were counted.In order to study the role of a specific protein in THALinjection, GFP expression increased significantly to 7.3 �

0.6 arbitrary units/mg protein (N � 4), peaking at 23.5 � function and physiology, it is necessary to selectively ex-press a transgene in THALs. We tested whether our4.9 arbitrary units/mg protein (N � 6) by 7 days. GFP

expression declined to 6.7 � 1.8 arbitrary units/mg pro- method was suitable for this by injecting a recombinantadenovirus in which GFP expression was driven by atein (N � 4) at 14 days postinjection, and was not signifi-

cantly different from background by 21 days (2.1 � 1.8 THAL-specific promoter. We used the promoter regionof the gene coding for the Na/K/2Cl cotransporter, Nkcc2arbitrary units/mg protein) (N � 4) (Fig. 6B). GFP ex-

pression was not detected in nontransduced kidneys. (which is only expressed in THAL cells) to drive GFP ex-pression [19, 22]. Only tubules of the outer medulla showedTaken together, these findings suggest that our technique

of delivering a viral vector to the kidney in vivo results GFP fluorescence. To make sure that all of these tubuleswere only THALs, we labeled Tamm-Horsfall proteinin efficient and transient transduction of THALs.with specific antibodies. We found that only tubules show-ing labeling for Tamm-Horsfall protein also expressed

DISCUSSIONGFP, whereas no fluorescence for GFP was detected in

To our knowledge, this is the first report of efficient nontargeted regions such as the cortex and inner medulla.adenovirus-mediated in vivo transduction of THAL cells. Thus, unlike gene transfer with Ad-CMVGFP, transduc-We found that injection of Ad-CMVGFP directly into tion with Ad-NKCC2GFP resulted in THAL-specific ex-the interstitium resulted in efficient transduction of all pression. When Ad-NKCC2GFP was injected into thecells in and near the injection track. In most studies cortex, cortical THAL cells could also be transduced.where viral vectors were infused either into the renal Thus, this promoter could also be used to target maculaartery [15, 16, 24] or retrograde through the ureter [13, 16], densa cells, which also express NKCC2, although trans-transduction efficiency of renal tubule cells has been low. duction efficiency would be lower than for medullaryIn some epithelial cells, the receptors needed for attach- THALs. Our data suggest that this method could alsoment of adenoviruses to the membrane have been localized be used to specifically transduce other nephron segmentsmainly to the basolateral membrane and tight junctions such as inner medullary collecting ducts or proximal tu-[25]. Thus, it is possible that the routes of administration bules by (1) adjusting the depth of injection to corre-used in those studies could have limited the number of spond to the desired target region, and (2) using cellrenal tubular cells exposed to the vector, resulting in low type–specific promoters to drive transgene expression.transduction efficiency. However, to our knowledge this Adenovirus-mediated gene transfer results in transienthas not been tested yet in renal epithelial cells. gene expression, due to the inability of the adenoviral

Clamping the renal artery and vein may prevent dilu- genome to become integrated into the host cell genome.tion and washout of viral particles from the interstitium Time-course experiments showed that GFP expressioninto the medullary circulation, allowing a higher number in THALs transduced with Ad-NKCC2GFP peaked byof viruses to interact with tubular cells and thereby in- 7 days postinjection and sharply declined by 14 days. Increasing transduction efficiency. We found no irreversible agreement with our data, Moullier et al [15] found thatpathologic changes in kidney structure after occluding expression of a reporter gene was also maximal 7 daysthe renal artery and vein for 8 minutes. In agreement after adenoviral transfection of the kidney in vivo. How-with the literature [16, 26–30], histologic or functional ever, different time-course expression has been reportedpathologic changes have only been observed after 15 in other tissues. Transduction of the carotid sinus showedminutes of renal ischemia and posterior reperfusion. a sharp increase in transgene expression by only 4 daysWhile some investigators have avoided renal ischemia [31]. Similarly, in transduced cardiac myocytes, transgeneduring viral delivery, others have found that this proce- expression peaked at 3 days postinjection and returneddure increases transduction efficiency [13, 16]. to basal levels by 7 to 14 days [32]. Possible reasons for

To increase exposure of THALs to the viral vectors, varying time-dependent expression of transgenes couldwe positioned the tip of the needle in the outer medulla. include (1) differences in the types of tissue transduced,By correlating body weight with the distance between (2) different transcriptional activities of the promotersthe capsule and the corticomedullary junction, the needle used to drive gene expression in a given tissue, and (3)was inserted deep enough that the virus was applied pri- different turnover rate of the proteins used as markers.marily to a specific region. This technique consistently Overall, our data demonstrate that our method foryielded a high transduction efficiency when the outer adenovirus-mediated gene transfer can be used to achievemedulla was targeted such that at least 80% of THAL ex- highly efficient in vivo transduction of kidney THALs.

Due to the transient gene expression observed and thepressed GFP. The confidence interval was less than �12%

Ortiz et al: In vivo transduction of THAL1148

insights from Bartter’s and Gitelman’s syndromes. Am J Physiolimpossibility for repeated injections of adenoviral vec-Renal Physiol 279:F616–F625, 2000tors, this technique is not likely to be used for human 6. Ito O, Roman RJ: Role of 20-HETE in elevating chloride transport

gene therapy. However, our method can be adapted to in the thick ascending limb of Dahl SS/Jr rats. Hypertension 33:419–423, 1999a wide variety of animal studies, and could provide an

7. Roman RJ, Kaldunski ML: Enhanced chloride reabsorption inimportant tool for investigators interested in dissecting the loop of Henle in Dahl salt-sensitive rats. Hypertension 17:1018–the molecular mechanisms involved in regulation of 1024, 1991

8. Garcia NH, Plato CF, Stoos BA, et al: Nitric oxide-inducednephron transport. Cell type–specific expression of dif-inhibition of transport by thick ascending limbs from Dahl salt-ferent genes can be achieved by adjusting the depth ofsensitive rats. Hypertension 34:508–513, 1999

injection and using cell-specific promoters. For example, 9. Plato CF, Shesely EG, Garvin JL: eNOS mediates l-arginine-induced inhibition of thick ascending limb chloride flux. Hyperten-a dominant negative mutant of a protein kinase can besion 35:319–323, 2000selectively expressed in collecting ducts under control of

10. Takahashi N, Brooks HL, Wade JB, et al: Posttranscriptionalthe aquaporin-2 promoter. Collecting ducts could then compensation for heterozygous disruption of the kidney-specific

NaK2Cl cotransporter gene. J Am Soc Nephrol 13:604–610, 2002be microdissected and the role of this kinase in the regu-11. Schnermann J: Sodium transport deficiency and sodium balancelation of sodium or water transport studied, something

in gene-targeted mice. Acta Physiol Scand 173:59–66, 2001previously possible only in cell culture. In addition, for 12. Heikkila P, Parpala T, Lukkarinen O, et al: Adenovirus-medi-investigations that require nonspecific transgene over- ated gene transfer into kidney glomeruli using an ex vivo and in

vivo kidney perfusion system—First steps towards gene therapyexpression, viral promoters can be used to transduce allof Alport syndrome. Gene Ther 3:21–27, 1996cells near the injection area. Our method allows us to 13. Zhu G, Nicolson AG, Cowley BD, et al: In vivo adenovirus-

study the role of a specific protein in regulation of THAL mediated gene transfer into normal and cystic rat kidneys. GeneTher 3:298–304, 1996function in vivo. Since transgene expression peaks by

14. Lipkowitz MS, Hanss B, Tulchin N, et al: Transduction of renal7 days after transfection, the contribution of a specificcells in vitro and in vivo by adeno-associated virus gene therapy

protein expressed in one tubular segment to whole-kid- vectors. J Am Soc Nephrol 10:1908–1915, 199915. Moullier P, Friedlander G, Calise D, et al: Adenoviral-mediatedney function may be studied. For example, an adult mouse

gene transfer to renal tubular cells in vivo. Kidney Int 45:1220–1225,can be generated in which the gene for cyclooxygenase-21994

(COX-2) is flanked by two loxP sequences. Then our 16. Chetboul V, Klonjkowski B, Lefebvre HP, et al: Short-termefficiency and safety of gene delivery into canine kidneys. Nephrolmethod can be used to transduce THALs with an adeno-Dial Transplant 16:608–614, 2001virus coding for Cre recombinase under control of the

17. Anderson RD, Haskell RE, Xia H, et al: A simple method forNKCC2 promoter. This maneuver would delete or invert the rapid generation of recombinant adenovirus vectors. Gene Therthe gene flanked by loxP, preventing its transduction 7:1034–1038, 2000

18. Flanagan SW, Anderson RD, Ross MA, et al: Overexpressionin THALs. Thus, one could generate a THAL-specificof manganese superoxide dismutase attenuates neuronal death inCOX-2 knockout mouse which could be used to deter- human cells expressing mutant (G37R) Cu/Zn-superoxide dismu-

mine whether this enzyme or its products may promote salt tase. J Neurochem 81:170–177, 200219. Igarashi P, Whyte DA, Li K, et al: Cloning and kidney cell-retention or excretion by modulating THAL function.

specific activity of the promoter of the murine renal Na�-K�-Clcotransporter gene. J Biol Chem 271:9666–9674, 1996

ACKNOWLEDGMENTS 20. Ortiz PA, Hong NJ, Garvin JL: NO decreases thick ascendinglimb chloride absorption by reducing Na�-K�-2Cl cotransporter

This work was supported in part by a grant from the National activity. Am J Physiol Renal Physiol 281:F819–F825, 2001Institutes of Health Heart, Lung and Blood Institute (HL 28982) to 21. Igarashi P, Vanden Heuvel GB, Payne JA, et al: Cloning, embry-Jeffrey L. Garvin. P. Ortiz was supported in part by a fellowship onic expression, and alternative splicing of a murine kidney-specific(0020438Z) from the American Heart Association. Na-K-Cl cotransporter. Am J Physiol 269:F405–F418, 1995

22. Gamba G, Miyanoshita A, Lombardi M, et al: Molecular cloning,Reprint requests to Jeffrey L. Garvin, Ph.D., Division of Hyperten- primary structure, and characterization of two members of the

sion and Vascular Research, Department of Internal Medicine, Henry mammalian electroneutral sodium-(potassium)-chloride cotrans-Ford Hospital, 2799 W. Grand Boulevard, Detroit, MI 48202, USA. porter family expressed in kidney. J Biol Chem 269:17713–17722,E-mail: jgarvin1@hfhs.org 1994

23. Yu H, Papa F, Sukhatme VP: Bovine and rodent Tamm-Horsfallprotein (THP) genes: Cloning, structural analysis, and promoterREFERENCESidentification. Gene Expr 4:63–75, 1994

1. Good DW: The thick ascending limb as a site of renal bicarbonate 24. Kitamura M, Taylor S, Unwin R, et al: Gene transfer into thereabsorption. Semin Nephrol 13:225–235, 1993 rat renal glomerulus via a mesangial cell vector: Site-specific deliv-

2. Greger R: Ion transport mechanisms in thick ascending limb of ery, in situ amplification, and sustained expression of an exogenousHenle’s loop of mammalian nephron. Physiol Rev 65:760–797, 1985 gene in vivo. J Clin Invest 94:497–505, 1994

3. Kunzelmann K, Hubner M, Vollmer M, et al: A Bartter’s syn- 25. Walters RW, Grunst T, Bergelson JM, et al: Basolateral localiza-drome mutation of ROMK1 exerts dominant negative effects on tion of fiber receptors limits adenovirus infection from the apicalK� conductance. Cell Physiol Biochem 10:117–124, 2000 surface of airway epithelia. J Biol Chem 274:10219–10226, 1999

4. Konrad M, Vollmer M, Lemmink HH, et al: Mutations in the 26. Nogae S, Miyazaki M, Kobayashi N, et al: Induction of apoptosischloride channel gene CLCNKB as a cause of classic Bartter syn- in ischemia-reperfusion model of mouse kidney: Possible involve-drome. J Am Soc Nephrol 11:1449–1459, 2000 ment of Fas. J Am Soc Nephrol 9:620–631, 1998

27. Defraigne JO, Pincemail J, Detry O, et al: Preservation of cortical5. Ellison DH: Divalent cation transport by the distal nephron:

Ortiz et al: In vivo transduction of THAL 1149

microcirculation after kidney ischemia-reperfusion: Value of an 30. Islam CF, Mathie RT, Dinneen MD, et al: Ischaemia-reperfusioninjury in the rat kidney: the effect of preconditioning. Br J Uroliron chelator. Ann Vasc Surg 8:457–467, 199479:842–847, 199728. Eschwege P, Paradis V, Conti M, et al: In situ detection of lipid

31. Meyrelles SS, Mao HZ, Heistad DD, et al: Gene transfer toperoxidation by-products as markers of renal ischemia injuries incarotid sinus in vivo: A novel approach to investigation of baro-

rat kidneys. J Urol 162:553–557, 1999 receptors. Hypertension 30:708–713, 199729. Toosy N, McMorris EL, Grace PA, et al: Ischaemic precondi- 32. Guzman RJ, Lemarchand P, Crystal RG, et al: Efficient gene

tioning protects the rat kidney from reperfusion injury. BJU Int transfer into myocardium by direct injection of adenovirus vectors.Circ Res 73:1202–1207, 199384:489–494, 1999