Upregulation of Na transporter abundances inresponse to chronic thiazide or lo - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:26 |只看该作者 |倒序浏览 |打印

作者：Ki YoungNa, Yoon KyuOh, Jin SukHan, Kwon WookJoo, Jung SangLee, Jae-HoEarm, Mark A.Knepper,  Gheun-HoKim作者单位：1 Department of Internal Medicine, Seoul NationalUniversity, Clinical Research Institute of Seoul National UniversityHospital, Department of Internal Medicine, Eulji MedicalCollege, Department of Internal Medicine, Hallym UniversityHangang Sacred Heart Hospital, Seoul, 110-744; Departmentof Internal . Z! ?1 u4 E) J, L1 S
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      【摘要】
   Furosemideand hydrochlorothiazide (HCTZ) exert their diuretic actions by bindingto apical Na transporters, viz., theNa -K -2Cl cotransporter inthe thick ascending limb and the Na -Cl cotransporter in the distal convoluted tubule, respectively. We carriedout semiquantitative immunoblotting and immunohistochemistry of ratkidneys to investigate whether chronic administration of furosemide orHCTZ is associated with compensatory changes in the abundance ofNa transporters downstream from the primary site ofaction. Osmotic minipumps were implanted into Sprague-Dawley rats todeliver furosemide (12 mg/day) or HCTZ (3.75 mg/day) for 7 days. Toprevent volume depletion, all animals were offered tap water and asolution containing 0.8% NaCl and 0.1% KCl as drinking fluid. Thediuretic/natriuretic response was quantified in response to both agentsby using quantitative urine collections. Semiquantitativeimmunoblotting revealed that the abundances of thick ascending limbNa -K -2Cl cotransporter and allthree subunits of the epithelial Na channel (ENaC) wereincreased by furosemide infusion. HCTZ infusion increased theabundances of thiazide-sensitive Na -Cl cotransporter and -ENaC in the cortex and - and -ENaC in the outer medulla. Consistent with these results, -ENaCimmunohistochemistry showed a remarkable increase in immunoreactivityin the principal cells of collecting ducts with either diuretictreatment. These increases in the abundance of Na transporters in response to chronic diuretic treatment may account forthe generation of diuretic tolerance associated with long-term diuretic use.
      【关键词】 collecting duct epithelial sodium channel distal convolutedtubule thiazidesensitive sodiumchloride cotransporter
   INTRODUCTION

DIURETICS ARE FREQUENTLY PRESCRIBED for treatment of hypertension and edematousdisorders. Although they are clinically useful to induce negativeNa balance, diuretic resistance is often encountered tolimit their further use. Diuretic resistance may be explained in partby increased Na absorption in downstream renal tubulesegments related to increased Na delivery( 10 ).

Furosemide and hydrochlorothiazide (HCTZ) are diuretics commonly usedin clinical practice. Furosemide exerts its diuretic action by bindingto the Na -K -2Cl cotransporter(NKCC2) in the thick ascending limb and blocking ion transport( 34 ). HCTZ exerts its diuretic action by binding to theNa -Cl cotransporter (NCC) in the distalconvoluted tubule ( 4 ). Increased Na deliveryto the distal nephron may result in enhanced Na absorptiondownstream from the distal site of diuretic action. Recent research inthe pathophysiology of other disorders of water and electrolyte balancesuggests that long-term adaptive mechanisms may be important in thesedisorders and may involve altered expression of transporter proteins,such as aquaporin water channels and Na transporters( 13, 19, 21, 22, 30 ). In this context, it appears possiblethat diuretic resistance produced by long-term diuretic administrationis in part due to adaptive increases in the abundances ofNa transporters in the distal nephron and collecting ducts./ A# b& O& p/ E4 ~
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Therefore, this study was undertaken to elucidate the molecular basisof the adaptive mechanisms in long-term use of diuretics. Wehypothesized that a secondary increase in distal delivery of Na may induce compensatory changes in the abundance ofNa transporters downstream from the primary site of thediuretic action. To test this hypothesis, we administered eitherfurosemide or HCTZ for 7 days to rats and investigated the effects onthe expression of Na transporter proteins by usingsemiquantitative immunoblotting and immunohistochemistry on tissue fromrat kidneys.6 C! P' F8 I6 Y) i; W
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METHODS. p" @' |" {% l

Animals and experimental protocols. Specific pathogen-free male Sprague-Dawley rats (170-230 g; SLC,Shizuoka, Japan) were placed in metabolism cages 3 days before thebeginning of the study. Control and treated rats were designated randomly, and all were provided with a daily, fixed amount of finelyground regular rat chow (18 g · 200 g bodywt 1 · day 1 ) and two separatebottles of drinking water, one containing 0.8% NaCl and 0.1% KCl andthe other containing tap water. All the rats ate all of the offered ratchow and showed a steady increase in body weight throughout the studyperiod. The chronic studies were carried out as follows.7 l- Z" @; o! L0 o& `' q
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Chronic furosemide infusion. Rats were anesthetized with enflurane (Choongwae Pharma, Seoul, Korea),and osmotic minipumps (model 2ML1, Alzet, Palo Alto, CA) weresubcutaneously implanted to deliver 12 mg/day of furosemide (Handok,Seoul, Korea). Furosemide was dissolved in a 1.7% ethanolamine solution. Control rats were implanted with the minipumps containing vehicle (ethanolamine) alone./ u0 S1 s  u/ I, e- Q" O$ w
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Chronic HCTZ infusion. Rats were infused with either 3.75 mg/day of HCTZ (YuHan, Seoul, Korea)or vehicle for 7 days. These infusions were achieved by using the samevehicle solution (1.7% ethanolamine) and osmotic minipumps asdescribed above.

Physiological measurements. During the course of the studies, daily urine was collected to evaluateresponses to the diuretics. Urine osmolality was measured with acryoscopic osmometer (Osmomat 030-D-M, Gonotec, Berlin, Germany), andurine electrolytes and creatinine were measured with an ion-selectivemethod (System E4A, Beckman Coulter, Fullerton, CA). Serum samples werecollected at the time each rat was killed for determination of theserum aldosterone concentration by radioimmunoassay (SPAC-S aldosteronekit, Daiichi Pharmaceutical, Tokyo, Japan).  r% z, A: V; u/ e: h# g2 ~5 z
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Semiquantitative immunoblotting. After 7 days of infusion, the rats were killed by decapitation,and kidneys were rapidly removed and placed in chilled isolation solution containing 250 mM sucrose, 10 mM triethanolamine (Sigma, St.Louis, MO), 1 µg/ml leupeptin (Sigma), and 0.1 mg/mlphenylmethylsulfonyl fluoride (Sigma) titrated to pH 7.6. Next, thekidneys were dissected to obtain cortex and inner stripe of the outermedulla. Each region was separately homogenized in 10 (cortex) or 1 ml(outer medulla) of ice-cold isolation solution at 15,000 rpm with threestrokes for 15 s with a tissue homogenizer (PowerGun 125, FisherScientific, Pittsburgh, PA). After homogenization, total proteinconcentration was measured by using the bicinchoninic acid proteinassay reagent kit (Sigma) and adjusted to 2 µg/µl with isolationsolution. The samples were then stabilized by the addition of 1 vol 5×Laemmli sample buffer/4 vol sample, heated to 60°C for 15 min.
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Initially, "loading gels" were done on each sample set to allowfine adjustment of loading amount to guarantee equal loading onsubsequent immunoblots. Five micrograms of protein from each samplewere loaded into each individual lane and electrophoresed on 12%polyacrylamide-SDS minigels by using Mini-PROTEAN III electophoresis apparatus (Bio-Rad, Hercules, CA) and then stained with Coomassie bluedye (0.025% solution made in 4.5% methanol and 1% acetic acid;G-250, Bio-Rad). Selected bands from these gels were scanned (GS-700Imaging Densitometry, Bio-Rad) to determine density (Molecular Analystversion 1.5, Bio-Rad) and relative amounts of protein loaded in eachlane. Finally, protein concentrations were "corrected" to reflectthese measurements. For immunoblotting, the proteins were transferredelectrophoretically from unstained gels to nitrocellulose membrane(Bio-Rad). After being blocked with 5% skim milk in PBS-T (80 mMNa 2 HPO 4, 20 mM NaH 2 PO 4,100 mM NaCl, and 0.1% Tween-20, pH 7.5) for 30 min, membranes wereprobed overnight at 4°C with the respective primary antibodies. Forprobing blots, all antibodies were diluted into a solution containing150 mM NaCl, 50 mM sodium phosphate, 10 mg/dl sodium azide, 50 mg/dlTween 20, and 0.1 g/dl bovine serum albumin (pH 7.5). The secondaryantibody was goat anti-rabbit IgG conjugated to horseradish peroxidase(31458, Pierce, Rockford, IL) diluted to 1:3,000. Sites ofantibody-antigen reaction were viewed by using enhancedchemiluminescence substrate (ECL RPN 2106, Amersham Pharmacia Biotech,Buckinghamshire, UK) before exposure to X-ray film (Hyperfilm, AmershamPharmacia Biotech).* u' k7 F. F% P) F, {' ~
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Immunohistochemistry. The kidneys were perfused by retrograde perfusion via the abdominalaorta with 1% PBS to remove blood and then withperiodate-lysine-paraformaldehyde (0.01 M NaIO 4, 0.075 Mlysine, and 2% paraformaldehyde in 0.0375 MNa 2 HPO 4 buffer, pH 6.2) for kidney fixation for3 min. After completion of perfusion, each kidney was sliced into5-mm-thick pieces and immersed in 2% periodate-lysine-paraformaldehydesolution overnight at 4°C. Each slice was dehydrated with a gradedseries of ethanol and embedded in polyester wax. The embedded pieces ofkidney were sectioned at 5-µm thickness on a microtome (RM 2145, Leica Instruments, Nussloch, Germany).

The sections were dewaxed with a graded series of ethanol and treatedwith 3% H 2 O 2 for 30 min to eliminateendogenous peroxidase activity. They were blocked with 6% normal goatserum (S-1000, Vector Laboratory, Burlington, CA) for 15 min. They wereincubated overnight at 4°C with the respective primary antibodiesdiluted in PBS. After incubation, they were washed with PBS andincubated for 30 min in biotinylated goat anti-rabbit IgG (BA-1000,Vector Laboratory) at room temperature. Next, peroxidase standardvectastatin ABC kit (PK-4000, Vector Laboratory) was added for30-60 min at room temperature. The sections were washed with PBSand incubated in 3,3'-diaminobenzidine substrate kit (SK-4100, VectorLaboratory). Hematoxylin staining was used as a counterstain. Theslides were mounted with Canadian balsam.  Y* x% _# p4 }) R, X; U. f# [

Primary antibodies. For semiquantitative immunoblotting and immunohistochemistry, we usedpreviously characterized polyclonal antibodies. Affinity-purified polyclonal antibodies against the thick ascending limb NKCC2( 9 ), thiazide-sensitive NCC ( 20 ), and -, -, and -subunits of the epithelial Na channel (ENaC)( 26 ) were used. In addition, the present study utilizedpolyclonal antibodies against aquaporin-1 (AQP1) ( 40 ), AQP2 ( 32 ), and AQP3 ( 8 ).# Q' J8 J' N3 e1 E
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Statistics. Values were presented as means ± SE. Quantitative comparisonsbetween the groups were made by Mann-Whitney U -test(Statview software, Abacus Concepts, Berkeley, CA). To facilitatecomparisons in the semiquantitative immunoblotting, we normalized theband density values by dividing by the mean value for the controlgroup. Thus the mean for the control group is defined as 100%. P was considered as indicative of statistical significance.& z! H; n) Q$ \  ?/ c
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RESULTS7 |/ M1 O2 ]. H

Chronic furosemide infusion. Physiological data from urine collections confirmed the effectsof furosemide. Table 1 shows urinarydata on the final day of chronic furosemide infusion. Urine output wasmarkedly increased and urine osmolality was decreased by furosemideinfusion compared with vehicle-infused controls. In furosemide-infusedrats, urinary Na and Cl excretion wasremarkably increased, but there was no significant change in urinaryK excretion. Creatinine clearance was also notsignificantly affected by furosemide infusion.
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Table 1. Urinary data from chronic furosemide infusion# T2 O% i  E& G1 {
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Despite persistent osmotic diuresis for 7 days, body weight measuredbefore euthanasia was not different between the two groups (furosemide-infused rats, 198 ± 4 g, vs. vehicle-infusedrats, 204 ± 4 g). Furthermore, serum aldosterone level wasnot altered by furosemide infusion (furosemide-infused rats, 0.40 ± 0.15 nM, vs. vehicle-infused rats, 0.38 ± 0.14 nM).3 z4 h1 `4 ?: V

Analysis of the renal cortical homogenates prepared for immunoblottingrevealed a significant increase in total amount of protein infurosemide-infused rat cortices compared with vehicle-infused controls(80.8 ± 3.7 vs. 66.0 ± 3.5 mg/100 g body wt, P This finding is consistent with thepreviously demonstrated furosemide-induced cortical hypertrophy( 17, 18 ). On the other hand, there was no difference intotal amount of protein in the outer medullary homogenates between thetwo groups." n: ^4 w$ t) x# Q3 L6 v2 F; l

Figure 1 A shows an immunoblotof the thick ascending limb NKCC2 from cortical homogenates.Furosemide infusion for 7 days resulted in a significant increase inNKCC2 abundance in the cortex. Normalized band densities forfurosemide-infused and vehicle-infused rats were 151 ± 10 and100 ± 10, respectively (means ± SE, P 0.05). In Fig. 1 C, the abundance of NKCC2 in the outermedulla was also increased by furosemide infusion (122 ± 5 forfurosemide-infused rats vs. 100 ± 3 for vehicle-infused rats, P Coomassie blue-stainedSDS-polyacrylamide gels demonstrated uniform loading among all samples(Fig. 1, B and D ), ruling out the possibility that the increase in band density in cortex or outer medulla could bedue to differences in loading.

Fig. 1. Effects of furosemide infusion on abundance of thickascending limb Na -K -2Cl cotransporter (NKCC2). Immunoblots of cortical homogenates( A ) and outer medullary homogenates ( C ) fromSprague-Dawley rats receiving a subcutaneous infusion of furosemide (12 mg/day) or vehicle via osmotic minipump are shown. Each lane was loadedwith protein sample from a different rat. Blots were probed with rabbitpolyclonal anti-NKCC2 antibody L224. A : blot of cortex wasloaded with 10 µg total protein/lane. Band density for NKCC2 proteinwas significantly increased by furosemide infusion. C : blotof outer medulla was loaded with 5 µg total protein/lane. Banddensity for NKCC2 protein was significantly increased by furosemideinfusion. Twelve percent Coomassie blue-stained SDS-polyacrylamide gelsfrom cortical homogenates ( B ) and outer medullaryhomogenates ( D ) confirm equal loading among lanes.

The abundance of thiazide-sensitive NCC in the cortex was not alteredby furosemide infusion (Fig. 2 ).Normalized band densities for furosemide-infused rats vs.vehicle-infused rats were 101 ± 11 vs. 100 ± 19, respectively. Figure 3 shows NCCimmunohistochemistry from the cortex of furosemide-infused andvehicle-infused rats. Consistent with previous studies ( 12, 17 ), distal tubular hypertrophy was noted in furosemide-infusedrats. However, no difference in NCC immunoreactivity was found betweenfurosemide-infused and vehicle-infused rats.4 B. E$ w$ y: M$ D3 F' E

Fig. 2. Effect of furosemide infusion on abundance ofthiazide-sensitive Na -Cl cotransporter (NCC)in cortex. Immunoblot of the same cortical homogenates fromSprague-Dawley rats as used for Fig. 1 is shown. Each lane wasloaded with a protein sample from a different rat. The blot was loadedwith 10 µg total protein/lane and was probed with rabbit polyclonalanti-NCC antibody L573. Band density for NCC protein was notsignificantly altered by furosemide infusion.3 X/ a, L9 B- A8 o
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Fig. 3. Immunohistochemistry of thiazide-sensitive NCC in cortex fromSprague-Dawley rats infused with vehicle or furosemide. Top,×100; bottom, ×400.4 H. N7 f" l, s
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Figure 4 shows the immunoblots carriedout for the three ENaC subunits in cortical homogenates. Significantincreases in all three subunits of ENaC protein abundance were found inresponse to chronic furosemide infusion. Normalized band densities ofeach subunit protein were as follows: -subunit (Fig. 4 A ),187 ± 25 for furosemide vs. 100 ± 22 for vehicle, P -subunit (Fig. 4 B ), 155 ± 8 for furosemide vs. 100 ± 15 for vehicle, P and -subunit (Fig. 4 C ) 168 ± 16 forfurosemide vs. 100 ± 9 for vehicle, P medulla of the same ratswere qualitatively the same as those seen in the cortex (Fig. 5 ). Band density of each subunit protein was as follows: -subunit (Fig. 5 A ), 171 ± 27 forfurosemide vs. 100 ± 17 for vehicle, P -subunit (Fig. 5 B ), 986 ± 91 for furosemide vs.100 ± 33% for vehicle, P -subunit (Fig. 5 C ), 242 ± 24 for furosemidevs. 100 ± 22 for vehicle, P

Fig. 4. Effects of furosemide infusion on abundance of epithelialNa channel (ENaC) in cortex. Each lane was loaded withcortical homogenate from a different rat. A : blot was loadedwith 30 µg total protein/lane and was probed with rabbit polyclonalanti- -ENaC antibody L766. B : blot was loaded with 30 µgtotal protein/lane and was probed with rabbit polyclonal anti- -ENaCantibody L558. C : blot was loaded with 30 µg totalprotein/lane and was probed with rabbit polyclonal anti- -ENaCantibody L550. Band density for each subunit protein was significantlyincreased by furosemide infusion.

Fig. 5. Effects of furosemide infusion on abundance of ENaC inouter medulla. Each lane was loaded with outer medullary homogenatefrom a different rat. A : blot was loaded with 30 µg totalprotein/lane and was probed with rabbit polyclonal anti- -ENaCantibody L766. B : blot was loaded with 30 µg totalprotein/lane and was probed with rabbit polyclonal anti- -ENaCantibody L558. C : blot was loaded with 30 µg totalprotein/lane and was probed with rabbit polyclonal anti- -ENaCantibody L550. Band density for each subunit protein was significantlyincreased by furosemide infusion.9 \0 [" v/ q2 |4 _

Figure 6 demonstrates ENaCimmunohistochemistry from the renal cortex of furosemide-infused andvehicle-infused rats by using the -ENaC antibody. Figure 6, top, shows low-power images demonstrating a marked increasein immunostaining in furosemide-infused vs. vehicle-infused rat. Inhigher-power images (Fig. 6, bottom ), the size of thetubular epithelial cells appeared larger in a furosemide-infused ratcompared with a vehicle-infused rat. Staining conditions and exposuresettings on the microscope were identical for the two images. Similarobservations were made in three pairs of rats.

Fig. 6. Immunohistochemistry of -ENaC in cortex fromSprague-Dawley rats infused with vehicle or furosemide. Each panelshows a marked increase in immunostaining in furosemide-infused vs.vehicle-infused rat. Top, ×100; bottom,×400.

We also carried out immunoblotting for AQP1-3 in the cortex andouter medulla (not shown). In the cortex, there were no significant changes in the band densities of AQP1 (105 ± 7 for furosemide vs.100 ± 7 for vehicle), AQP2 (149 ± 18 for furosemide vs.100 ± 17 for vehicle), and AQP3 (126 ± 17 for furosemidevs. 100 ± 12 for vehicle). As in the cortex, band density foreach aquaporin protein in the outer medulla was not significantlyaltered by furosemide infusion (AQP1, 80 ± 9 vs. 100 ± 9;AQP2, 94 ± 11 vs. 100 ± 7; and AQP3, 72 ± 12 vs. 100 ± 22).
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Chronic HCTZ infusion. Chronic infusion of HCTZ revealed similar changes in urinary indices tofurosemide infusion. Table 2 showsurinary data on the final day of HCTZ infusion. HCTZ produced asignificant increase in urine output and a decrease in urineosmolality. As in the furosemide infusion study, urinaryNa and Cl excretion were elevated butK excretion was not significantly increased inHCTZ-infused rats. Creatinine clearance was not affected.& A, M9 {/ S8 I6 w" e
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Table 2. Urinary data from chronic hydrochlorothiazide infusion0 G& Q: ?7 d: N
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In the HCTZ infusion study, the total amount of cortical proteinmeasured in the homogenates of cortex dissected from the kidneys ofHCTZ-infused rats was not different from the corresponding value ofvehicle-infused rats (68.6 ± 2.4 vs. 65.2 ± 1.5 mg/100 gbody wt). The two groups had no difference in total amount of proteinin the outer medullary homogenates.
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Figure 7 A shows an NCCimmunoblot in cortical homogenates from vehicle-infused andHCTZ-infused rats. The abundance of NCC protein in HCTZ-infused ratswas significantly increased compared with vehicle-infused rats(normalized band densities: 166 ± 14 for HCTZ vs. 100 ± 13 for vehicle, P 7 B shows aloading control for the immunoblot, achieved by running an identically loaded polyacrylamide gel and staining it with Coomassie blue. Therewas little or no variation in the densities of the major bands stainedwith Coomassie blue, demonstrating that the increase in NCC banddensity seen in Fig. 7 A was not due to unequal loading.
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Fig. 7. Effect of hydrochlorothiazide (HCTZ) infusion onabundance of thiazide-sensitive NCC in cortex. Immunoblot of corticalhomogenates from Sprague-Dawley rats receiving a subcutaneous infusionof HCTZ (3.75 mg/day) or vehicle via osmotic minipump is shown. Eachlane was loaded with protein sample from a different rat. A : blot was loaded with 10 µg total protein/lane and wasprobed with rabbit polyclonal anti-NCC antibody L573. Band density forNCC protein was significantly increased by HCTZ infusion. B :12% Coomassie blue-stained SDS-polyacrylamide gel from corticalhomogenates, confirming equal loading among lanes.2 M! s4 k! u5 G# M& G& J

Figure 8 shows NCC immunohistochemistryfrom the cortex of HCTZ-infused and vehicle-infused rats. Compatiblewith the immunoblot result, it reveals an increase in immunostaining inan HCTZ-infused vs. a vehicle-infused rat. Similar observations weremade in three pairs of rats." ]  H7 b  u/ l$ r
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Fig. 8. Immunohistochemistry of thiazide-sensitive NCC in cortex fromSprague-Dawley rats infused with vehicle or HCTZ. An increase inimmunostaining is noted in HCTZ-infused vs. vehicle-infused rat. Top, ×100; bottom, ×400.
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Figure 9 shows the immunoblots from thesame cortical homogenates for the three ENaC subunits. In response toHCTZ infusion, an increase in -ENaC protein abundance was noted(Fig. 9 B ). Normalized band density increased to 139 ± 7 of vehicle-infused controls (100 ± 4, P in the abundance ofthe -subunit (Fig. 9 A; 111 ± 13 for HCTZ vs.100 ± 11 for vehicle) or -subunit (Fig. 9 C;122 ± 10 for HCTZ vs. 100 ± 12 for vehicle). Changes in theabundance of ENaC in the outer medulla are depicted in Fig. 10. Band density of -ENaC was notsignificantly increased by HCTZ infusion (Fig. 10 A; 122 ± 19 for HCTZ vs. 100 ± 9 for vehicle). In contrast, both -ENaC (Fig. 10 B; 201 ± 28 for HCTZ vs. 100 ± 16 for vehicle, P and -ENaC (Fig. 10 C; 212 ± 17 for HCTZ vs. 100 ± 8 for vehicle, P homogenates. A parallel Coomassie blue-stainedSDS-polyacrylamide gel demonstrated uniform loading among all samples(Fig. 10 D ), ruling out the possibility that the increase inband density in the outer medulla could be due to differences inloading.

Fig. 9. Effects of HCTZ infusion on abundance of ENaC in cortex.Each lane was loaded with cortical homogenate from a different rat. A : blot was loaded with 30 µg total protein/lane and wasprobed with rabbit polyclonal anti- -ENaC antibody L766. Band densityfor -ENaC protein was not significantly altered by HCTZ infusion. B : blot was loaded with 30 µg total protein/lane and wasprobed with rabbit polyclonal anti- -ENaC antibody L558. Band densityfor -ENaC protein was significantly increased by HCTZ infusion. C : blot was loaded with 30 µg total protein/lane and wasprobed with rabbit polyclonal anti- -ENaC antibody L550. Band densityfor -ENaC protein was not significantly altered by HCTZ infusion.. G+ r6 a3 S$ m5 L8 c+ K/ W
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Fig. 10. Effects of HCTZ infusion on abundance of ENaC in outermedulla. Immunoblots of outer medullary homogenates from Sprague-Dawleyrats receiving a subcutaneous infusion of HCTZ (3.75 mg/day) or vehiclevia osmotic minipump are shown. Each lane was loaded with proteinsample from a different rat. A : blot was loaded with 30 µgtotal protein/lane and was probed with rabbit polyclonal anti- -ENaCantibody L766. Band density for -ENaC protein was not significantlyaltered by HCTZ infusion. B : blot was loaded with 30 µgtotal protein/lane and was probed with rabbit polyclonal anti- -ENaCantibody L558. Band density for -ENaC protein was significantlyincreased by HCTZ infusion. C : blot was loaded with 30 µgtotal protein/lane and was probed with rabbit polyclonal anti- -ENaCantibody L550. Band density for -ENaC protein was significantlyincreased by HCTZ infusion. D : 12% Coomassie blue-stainedSDS-polyacrylamide gel from outer medulla, confirming equal loadingamong lanes.
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Figure 11 demonstrates ENaCimmunohistochemistry from the cortex of HCTZ-infused andvehicle-infused rats by using the -ENaC antibody. Compatible withthe immunoblot result, it shows an increase in immunostaining in anHCTZ-infused vs. a vehicle-infused rat. Similar observations were madein three pairs of rats.% X& o- R% p  d! v* Z7 W

Fig. 11. Immunohistochemistry of -ENaC in cortex fromSprague- Dawley rats infused with vehicle or HCTZ. Each panelshows an increase in immunostaining in HCTZ-infused vs. vehicle-infusedrat. Top, ×100; bottom, ×400.

DISCUSSION! L: r* O& t4 Q$ g) @/ {. q# f

In this study, we analyzed changes in the abundance of the majorapical Na transporters in the distal sites of the renaltubule in response to chronic infusion of a loop diuretic (furosemide)or a thiazide diuretic (HCTZ) in rats. Table 3 summarizes the percent changes inNKCC2, NCC, and three subunits of ENaC protein abundances with eitherdiuretic treatment. The data provide important new information relevantto clinical use of these diuretics and address an important physiological issue: What is the effect of increased distal fluid delivery on transporter abundances? The main results showed that theNa channel expressed in connecting tubule and collectingduct, ENaC, was increased in its abundance in response to chronicinfusion of either diuretic, consistent with previous reports showingincreased Na absorption in these segments ( 14, 38 ). In addition, furosemide induced an increased abundance ofNKCC2, whereas HCTZ induced an increased abundance of NCC. Theimportant clinical implication from our study is that the diuretictolerance associated with long-term diuretic use may be caused byincreases in the abundance of distal Na transporters.; L" F% R& s8 i
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Table 3. Percent changes in Na transporter abundances in responseto chronic furosemide or hydrochlorothiazide infusion

Chronic diuretic infusion induces compensatory increase in ENaCabundance. Both furosemide and HCTZ caused a substantial diuresis and natriuresis,although the response was greater with furosemide than with HCTZ. Withconcomitant salt and water substitution, the animals gained body weightsteadily without apparent volume depletion during the experiment. Onthe basis of the known sites of action of the diuretics, it is likelythat Na delivery to collecting duct was increased by both diuretics.

ENaC constitutes the rate-limiting step for Na reabsorption in the connecting tubule and collecting duct in the ratkidney ( 6, 16, 36 ). Because ENaC is a heteromultimericprotein formed by the association of three homologous subunits,,, and ( 2 ), we tested its expression by using thespecific polyclonal antibodies to each subunit ( 26 ). Inour study, chronic furosemide infusion increased the protein abundanceof all three subunits in the cortex and more prominently in the outermedulla. Chronic HCTZ infusion also increased the abundance of -subunit in the cortex and - and -subunits in the outermedulla. Although regulation of ENaC activity in the collecting ductand connecting tubule involves several control processes, such asregulated trafficking ( 16, 25 ), and posttranslationalmodifications, such as methylation ( 34 ), it is likely thatan important element of ENaC regulation is achieved through control ofsubunit abundance as seen in response to aldosterone or vasopressin( 7, 26 ). We did not investigate the mechanism of alteredENaC subunit protein abundance in the present study, althoughregulation of mRNA levels ( 31, 39 ) and regulation of ENaCprotein half-life ( 5, 27, 41 ) have been described inprevious studies.

Our animal protocol of salt and water substitution was apparentlyeffective in preventing extracellular fluid volume depletion, asevidenced by the absence of an increase in serum aldosterone level.Furthermore, NCC abundance, which is regulated by aldosterone ( 20 ), was not altered by chronic furosemideadministration. Thus it appears unlikely that the changes in ENaCsubunit abundance are mediated by either aldosterone or vasopressin.Another factor previously shown to alter ENaC subunit abundance isacid-base state. Alkali loading was demonstrated to increase both -and -ENaC abundance in rat kidney ( 19 ), raising thepossibility that the increases in the abundance of these two subunitscould be related to the metabolic alkalosis seen with chronic diuretic adminstration.
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On the basis of prior literature, it appears quite plausible that theincrease in ENaC subunit expression seen with chronic diureticadministration is related to the effects on the delivery of NaCl andwater to the collecting duct. Specifically, in furosemide-infused, volume-replaced rats, Stanton and Kaissling ( 18, 38 )demonstrated an increase in distal Na absorptionaccompanied by an increase in epithelial cell volume of the distalconvoluted tubule, connecting tubule, and collecting duct. Consistentwith a general hypertropic effect of chronic furosemide infusion, wefound that the total amount of protein in cortical homogenates waslarger in furosemide-infused rats compared with vehicle-infusedcontrols. These findings seem to be compatible with a previous study( 23 ) showing that furosemide infusion with high-saltintake caused an increase in DNA synthesis in distal segmentsdownstream of the thick ascending limb. The mechanism by whichthe renal tubule cells sense an increase in flow rate is unknown butmay involve the central cilium present in renal tubule epithelial cells( 33 ).
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An increase in Na -K -ATPase activity in thecortical collecting duct was shown after either furosemide( 37 ) or HCTZ ( 14 ) administration. Althoughthe mechanism is unknown, the increase in basolateralNa -K -ATPase activity would reduceintracellular Na  , promoting Na influx viaapical ENaC in the collecting duct. A regulatory role of alteredintracellular and extracellular ion concentrations on ENaC function hasbeen reviewed ( 15 ), and it appears likely thatintracellular Na activity has effects on ENaC that areindependent of effects on the electrochemical driving forces forNa ion movement through the channel. These effects couldpossibly include altered abundance of ENaC subunit proteins. Althoughchanges in transporter abundance are by no means the only way in which transport capacity of renal tubule cells is regulated, the increase inENaC abundance that we have observed is likely to represent adaptiveresponses to allow for the conservation of Na in responseto diuretic administration.$ ]3 F, d: a% _+ d2 v# A( u

Abundance of NKCC2 and NCC is increased by furosemide and HCTZinfusion, respectively. Chronic furosemide infusion significantly increased the abundanceof NKCC2 in cortex and outer medulla, and chronic HCTZ infusion markedly increased the abundance of NCC in cortex. This may represent acompensatory response and may contribute to diuretic resistance. Consistent with the effect of HCTZ administration on NCC abundance, aprevious study demonstrated that the binding density of[ 3 H]metolazone, an indirect measure of NCC abundance, wasincreased by chronic HCTZ infusion ( 3, 29 ) despite adecrease in NaCl transport capacity of distal convoluted tubule( 11 ). The distal convoluted tubules from HCTZ-infused ratspreserved epithelial structure and unipolar apical staining when theywere examined by the NCC immunohistochemistry (Fig. 8 ). These findingsare in contrast to the report of Loffing et al. ( 24 ) thatthiazide (especially metolazone) treatment in Wistar rats wasassociated with injury of distal convoluted tubule cells, a basal shiftof NCC localization, and a significant decrease in mRNA transcripts ofNCC ( 24 ). We do not know whether the discrepancies result from differences in animals (Sprague-Dawley vs. Wistar rats) and doses(3.75 mg·rat 1 ·day 1 in the present study vs. 40 mg·kg bodywt 1 ·day 1 ) of HCTZ.

Another interesting finding is that NCC abundance was not increased byfurosemide infusion. Previous micropuncture experiments in rat kidneyshave demonstrated that chronic furosemide infusion increasesNa reabsorption in the distal tubule ( 12, 38 ), an effect that is perhaps related to increased transportvia ENaC. However, in rats, chronic furosemide infusion increasedbinding of [ 3 H]metolazone to renal cortical membranes,suggesting an increase in NCC abundance ( 3 ). Recently,Abdallah et al. ( 1 ) reported that furosemide infusionincreased NCC protein abundance, but this effect was blocked byspironolactone, suggesting that the response was dependent on a rise inplasma aldosterone concentration. Compared with our study, they usedfurosemide in much larger doses (125 mg· kg 1 ·day 1 ),leading to a 10-fold increase in daily urine volume. Our finding thatNCC abundance was not increased by furosemide infusion may be relatedto the lack of an increase in serum aldosterone in our study. Inaddition, other neurohormonal factors can be considered as contributingto the changes in NCC protein abundance during furosemide infusion.Prostaglandin E 2 production can be stimulated by furosemideadministration ( 28 ), and nitric oxide generation can beincreased by high-salt intake ( 42 ). The possibility that these factors might act to affect NCC protein abundance needs to beinvestigated in future studies.

ACKNOWLEDGEMENTS; f, @. z: X& K5 k

The authors thank So-Young Kim, Han-Na Jung, and Prof. Seoung-WanChae for technical assistance.& U/ o' ?6 W1 S! M7 r  H1 X
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