Responses of proximal tubule sodium transporters to acuteinjury-induced hyperte - 《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 13:32 |只看该作者 |倒序浏览 |打印

作者：Li E.Yang, Patrick K. K.Leong, ShaohuaYe, Vito M.Campese,  Alicia A.McDonough作者单位：1 Department of Physiology and Biophysics, Division of Nephrology, Department of Medicine,University of Southern California Keck School of Medicine, Los Angeles,California 90089-9142



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      【摘要】
   Renal injury-induced byphenol injection activates renal sympathetic afferent pathways,increases norepinephrine release from the posterior hypothalamus,activates renal efferent pathways, and provokes a rapid and persistenthypertension. This study aimed to determine whether phenol injuryprovoked a redistribution of proximal Na transporters frominternal stores to the apical cell surface mediated by sympatheticactivation, a response that could contribute to generation ormaintenance of hypertension. Anesthetized rats were cannulated forarterial blood pressure tracing and saline infusion and then 50 µl10% phenol or saline was injected into one renal cortex( n = 7 each). Fifty minutes after injection, kidneyswere removed and renal cortex membranes from injected kidneys werefractionated on sorbitol gradients and pooled into three windows(WI-WIII) that contained enriched apical brush border (WI); mixedapical, intermicrovillar cleft and dense apical tubules (WII); andintracellular membranes (WIII). Na transporterdistributions were determined by immunoblot and expressed as percentageof total in gradient. Acute phenol injury increased blood pressure20-30 mmHg and led to redistribution ofNa /H exchanger type 3 (NHE3) out of WIII(from 22.79 ± 4.75 to 10.79 ± 2.01% of total) to WI(13.07 ± 1.97 to 27.15 ± 4.08%),Na -P i cotransporter 2 out of WII (68.72 ± 1.95 to 59.76 ± 2.21%) into WI (9.5 ± 1.62 to18.7 ± 1.45%), and a similar realignment of dipeptidyl-peptidaseIV immunoreactivity and alkaline phosphatase activity to WI. Renaldenervation before phenol injection prevented the NHE3redistribution. By confocal microscopy, NHE3 localized tothe brush border after phenol injection. The results indicate thatphenol injury provokes redistribution of Na transportersfrom intermicrovillar cleft/intracellular membrane pools toapical membranes associated with sympathetic nervous system activation,which may contribute to phenol injury-induced hypertension. / h9 ?9 U" k5 i4 k$ }( Q
      【关键词】 kidney sympathetic nervous system phenol sodium/hydrogen exchanger type* K% y8 i; u! I
   INTRODUCTION" a$ Q0 g2 S8 ^% F( b" l

A RAT MODEL OF NEUROGENIC hypertension provoked by a renal injury was recentlydeveloped by Campese and colleagues ( 10, 43, 44 ). In thismodel, injection of 50 µl 10% phenol causes a rapid elevation ofblood pressure, which is preceded by a rise in norepinephrine secretionfrom the posterior hypothalamus and an increase in renal sympatheticnervous system (SNS) activity. Renal denervation before the renalinjury prevents the SNS activation and the subsequent rise in bloodpressure ( 44 ). Thus renal injury activates renal afferentpathways, increases norepinephrine release from the posteriorhypothalamus, activates renal efferent pathways, and raises bloodpressure. Interestingly, the hypertension becomes established andpersists long after the site of injury recedes to a microscopic scarand is reversed by removal of the injured kidney or renal denervation( 43 ). The cellular and molecular bases for thehypertension are not understood. One plausible mechanism is that therenal efferent sympathetic nerve activity may stimulate Na and volume reabsorption and contribute to hypertension.
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There is a dynamic relationship between blood pressure and renalNa reabsorption that is responsible for the blood pressureset point. A decrease in Na transport can be a homeostaticcompensation to elevated blood pressure; an experimental increase inblood pressure acutely decreases proximal tubule Na reabsorption, which both increases NaCl at the macula densa, atransforming growth factor signal to normalize renal blood flow (RBF)and glomerular filtration rate (GFR), and causes a pressure natriuresisthat reduces extracellular volume, which counteracts the hypertension( 8, 13, 14 ). In contrast, inappropriately elevatedNa transport, due to either excess production of anantinatriuretic substance (e.g., aldosterone) ( 40 ) or anactivated Na transporter (Liddle's syndrome)( 33 ), can generate and maintain hypertension. Bothresponses can occur together; if renal Na reabsorption iselevated, then blood pressure increases and induces apressure-natriuresis variant known as "escape" in whichNa reabsorption is depressed at sites along the nephronnot primarily affected by the excess hormone or Na transport, a response that balances Na excretion toNa intake.: ]( E5 q( Z8 I! _3 A
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Our laboratory previously investigated the molecular mechanismsresponsible for the decrease in proximal tubule Na reabsorption during an experimental 5-min increase in blood pressure and discovered that there is a rapid retraction ofNa /H exchangers[Na /H exchanger type 3 (NHE3)] andNa -P i cotransporters (NaPi) from the apicalbrush border to intermicrovillar cleft and subapical endosomes,demonstrated by both subcellular fractionation and confocal microscopy,as well as a decrease in basolateralNa -K -ATPase activity ( 42, 47 ).Motivated by these findings, we aimed to test the hypothesis that acutephenol injury, via activation of sympathetic efferents, increasesproximal tubule Na transport by recruiting Na transporters from subapical pools to the brush border, contributing tothe genesis of the hypertension. There is support for this hypothesisfrom in vitro studies on adrenergic regulation of proximal Na transporters ( 3, 4, 34 ). The results ofthis study support the hypothesis that acute phenol injury provokesredistribution of proximal tubule NHE3 from subapical endosomal poolsto apical brush border and that the response is mediated by sympathetic stimulation and may contribute to the generation and persistence ofphenol injury-induced hypertension.2 z( v3 r) E  R7 G9 {# R' s2 P

METHODS

Animal preparation. Experiments were performed on male Sprague-Dawley rats (300-350 gbody wt) that had free access to food and water before the experiment.Rats were anesthetized intramuscularly with ketamine (Fort DodgeLaboratories) and xylazine (1:1, vol/vol; Miles) and then placed on athermostatically controlled warming table to maintain body temperatureat 37°C. A polyethylene catheter was placed into the carotid arteryto monitor blood pressure. The jugular vein was cannulated to infuse4.0% BSA in 0.9% NaCl at 50 µl/min throughout the entireexperimental period to maintain euvolemia. The left side ureter wascannulated to collect urine.( I7 L& N) [9 }

The experimental time course was as follows. Blood pressure wasmeasured continuously. A baseline urine sample was collected over 20 min, and then 50 µl of 10% phenol or saline (sham control) wasinjected into the cortical lower pole of the left kidney( n = 7 in each group). Thirty minutes after injection,another urine sample was collected over 20 min, and then kidneys wereremoved for immediate analysis. On the same day, a phenol-injectedanimal was analyzed in parallel with a saline-injected animal andanalyzed in a paired fashion. In two sets, the effects of denervationon the response to phenol or saline injection was assessed. The left renal nerve was isolated by dissection and removed, and blood pressurewas allowed to stabilize for at least 30 min before phenol injection asdescribed by Ye et al. ( 44 ).8 d& }8 q$ H0 s: G- `  h
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For the experiments comparing arterial constriction hypertension tophenol injury-induced hypertension, mean arterial pressure wasincreased 20-30 mmHg by constricting the superior mesenteric artery, celiac artery, and abdominal aorta below the renal artery bytying silk ligatures around the vessels, as reported previously ( 46 ).
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Urine collection and endogenous lithium clearance. Urine was collected from the left ureter catheter, and urinary volumewas determined gravimetrically. A blood sample was collected after thekidneys were removed. The concentrations of endogenous lithium in bloodand urine samples were measured by flameless atomic absorptionspectrophotometry (Perkin-Elmer 5100PC) as described previously( 47 ).
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Homogenization and subcellular fractionation. In preliminary fractionation experiments, the injected and thecontralateral kidneys were compared, and the results wereindistinguishable. The injected kidneys were chosen for analysis.The procedure for subcellular fractionation of renal cortex membraneshas been described previously ( 46, 47 ). In brief, theinjected kidney from each phenol- or saline-injected animal was cooledin situ by flushing with cold PBS and then excised. The renal cortexwas dissected (injured area, ~2-mm diameter, was cut off anddiscarded), homogenized with a Tissuemizer (Tekmar Instruments) inisolation buffer [5% sorbitol, 0.5 mM disodium EDTA, 0.2 mMphenylmethylsulfonyl fluoride, 9 µg/ml aprotinin, and 5 mMhistidine-imidazole buffer (pH 7.5)], and centrifuged at 2,000 g for 10 min; the pellet was rehomogenized and centrifuged;and the low-speed supernatants were pooled, loaded between twohyperbolic sorbitol gradients, and centrifuged at 100,000 g for 5 h. Twelve fractions were collected from the top, diluted with isolation buffer, pelleted by centrifugation (250,000 g for 1.5 h), resuspended in 1 ml isolation buffer, andstored at 80°C, pending assays.

Immunoblot analysis and antibodies. A constant volume of sample from each gradient fraction was denaturedin SDS-PAGE sample buffer for 30 min at 37°C, resolved on the same7.5% SDS polyacrylamide gel according to Laemmli ( 20 ), and transferred to polyvinylidene difluoride membranes (Millipore Immobilon-P). Selected samples on each blot were run at one-half thevolume or protein to assure that the sample was in the linear range ofdetection, and multiple exposures of autoradiograms were analyzed toensure that signals were within the linear range of the film. Allblots, except for NaPi2 analysis, were detected with the ECL enhancedchemiluminescence kit (Amersham Pharmacia Biotech), andautoradiographic signals were quantified with a Bio-Rad imagingdensitometer with Molecular Analyst software. For NHE3 detection, blotswere probed with polyclonal NHE3-C00 ( 42 ) at 1:1,000dilution. Polyclonal antisera to dipeptidyl-peptidase IV (DPPIV) weregenerously provided by M. Farquhar (University of California at SanDiego). For detection of Na-P i cotransporter 2 (NaPi2),blots were incubated with polyclonal anti-NaPi2 antibody generated byBiber and Murer (University of Zürich, Zurich, Switzerland), thenwith Alexa 680-labeled goat-anti-rabbit secondary antibody, and thendetected with an Odyssey Infrared Imaging System (LI-COR, Lincoln, NB).Each gradient and immunoblot was from a separate rat, i.e., no poolingof samples. Results were quantitated by normalizing the density in eachfraction to the total sum density from all the fractions and expressedas the percentage of total immunoreactivity within each sample. Thesefraction-specific results were pooled into three "windows" tosimplify analyses. As previously reported ( 42, 46 ), fractions 3-5 [window I (WI)] are enriched in apicalbrush-border markers alkaline phosphatase, DPPIV, and NHE3; fractions 6-8 (WII) contain most of theintermicrovillar cleft marker megalin ( 5 ) as well asapical markers; and fractions 9-11 (WIII) are enrichedin the endosomal marker rab 5a and the lysosomal marker -hexosaminidase as well as megalin.
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Indirect immunofluorescence. To compare the NHE3 distribution in two models of acute hypertension(arterial constriction vs. phenol injury), blood pressure was raised tothe same level in each model (20-30 mmHg over baseline asdescribed above), and then one kidney from each animal was analyzed byconfocal microscopy. The kidney contralateral to the phenol or salineinjection was analyzed in this series to focus on the effects of thesimilarly elevated blood pressure in the two models and to eliminateany potential effect of the phenol per se. Kidneys were fixed at30-50 min in situ by placing the isolated kidney in a smallplexiglass cup and bathing it in fixative [2% paraformaldehyde, 75 mMlysine, and 10 mM Na-periodate, pH 7.4 (PLP)] for 20 min. The kidneyswere then removed, cut in half on a midsagittal plane, and postfixed inPLP for another 4-6 h. The fixed tissue was rinsed twice with PBS,cryoprotected by incubation overnight in 30% sucrose in PBS, embeddedin Tissue-Tek OCT Compound (Sakura Finetek, Torrance, CA), and frozenin liquid nitrogen. Cryosections (5 µM) were cut with a MicromHeidelgerg ultramicrotome, transferred to Fisher SuperfrostPlus-charged glass slides, and air dried. For immunofluorescencelabeling, the sections were rehydrated in PBS for 10 min, followed by10-min washing with 50 mM NH 4 Cl in PBS and then with 1%SDS in PBS for 4 min for antigen retrieval ( 9 ). SDS wasremoved by two 5-min washes in PBS, and then sections were blocked with1% BSA in PBS to reduce background. Double labeling was performed byincubating with polyclonal antiserum NHE3-C00 and monoclonal antibodyagainst villin (Immunotech, Chicago, IL), both at a dilution of 1:100in 1% BSA in PBS for 1.5 h at room temperature. After beingwashed for 5 min three times in PBS, the sections were incubated with amixture of FITC-conjugated goat-anti-rabbit (Cappel Research Products,Durham, NC) and Alexa 568-conjugated goat-anti-mouse (MolecularProbes, Eugene, OR) secondary antibodies diluted 1:100 in 1% BSAin PBS for 1 h, washed three times with PBS, mounted in ProlongAntifade (Molecular Probes), and dried overnight at room temperature.Slides were viewed with a Nikon PCM quantitative measuringhigh-performance confocal system equipped with filters for both FITCand tetramethylrhodamine isothiocyanate fluorescence attached to aNikon TE300 Quantum upright microscope. Images were acquired withSimple PCI C-Imaging hardware and Quantitative Measuring software andprocessed with Adobe PhotoDeluxe (Adobe Systems, Mountain View, CA).: |2 ^+ r! s- t% G! {5 r5 ]; [+ B3 j9 k

Other assays. Na -K -ATPase activity was measured by theK -dependent p -nitrophenyl phosphatase reaction( 30 ). Standard assay was used for alkaline phosphataseactivity ( 29 ). Protein concentrations were measured with abicinchoninic acid assay kit (Pierce Technology, Iselin, NJ).) w" r5 D; G& T
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Quantitation and statistical analysis. Experiments were conducted and analyzed in a pairwise fashion, that is,one phenol injected and one saline injected in 1 day. Data areexpressed as means ± SE. Two-way ANOVA was applied to determinewhether there was a significant effect of treatment on the overallsubcellular distribution pattern. If a significance was established( P thepattern was assessed by two-tailed Student's t -test forpaired samples, and the differences were regarded significant at P 0.05.
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RESULTS
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Physiological responses to acute phenol injury. Ye et al. ( 43, 44 ) reported that acute renal injury by anintrarenal injection of 50 µl 10% phenol caused an immediate andpermanent elevation in blood pressure. In this study, we verify thatthe effect of phenol injury could be reproduced in our laboratory withour animal preparation. Arterial blood pressure increased immediatelyafter 50 µl of 10% phenol injection, fluctuated somewhat over thenext 30 min, and then was maintained at 20-30 mmHg above baseline, an average increase from 110 ± 2.8 to 134 ± 2.4 mmHg (Fig. 1 ). These arterialpressures are within the autoregulatory range for GFR and RBF( 13 ). Sham injection of 50 µl of saline caused a brieftransient fluctuation in blood pressure that returned to baselinewithin 30 min (Fig. 1 A, bottom ).

Fig. 1. Physiological responses to acute phenol injury. A : representative blood pressure traces before and afterphenol ( top ) or saline ( bottom ) injection. B : summary of systolic arterial pressure before and 30 minafter injection, recorded from carotid artery. BP, blood pressure.Values are means ± SE; n = 7 for each group.* P t -test. C : urine output collected over 20-minintervals before and after 30-min phenol injection, expressed as urineweight in µg/min. D : endogeneous lithium clearance(C Li ) calculated as urinary lithium concentration × urine output × plasma lithium concentration 1 (µl/min)., Data from individual rats;, mean values; n = 9 for each group.. p" A! t1 \/ ?) H
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For each animal, urine was collected for 20 min before the phenol orsaline injection and for 20 min between 30 and 50 min after theinjection. Figure 1, C and D, summarizes theeffects of intrarenal phenol injection on urine output and endogenous lithium clearance (C Li ), a measure of volume flow from theproximal tubule ( 39 ). The mean values were elevatedslightly, although not statistically significantly: mean urine outputwas 10.3 ± 3.3 before and 18.5 ± 3.0 µg/min 30-50min after phenol injection and C Li was 60.65 ± 11.1 before and 95.12 ± 12.8 µl/min after phenol injection. Incomparison, acutely raising blood pressure by arterial constrictioncauses a marked pressure diuresis with consistent three- to fourfoldincreases in C Li and urinary output ( 42, 47 ).

NHE3 redistribution in response to phenol injury. Na /H exchanger is the major route for apicalNa entry across the proximal tubule, and NHE3 isresponsible for virtually all the Na /H exchange activity in this region ( 2, 6 ). Our previousstudies using confocal microscopy established that acute hypertension due to arterial constriction is associated with a rapid redistribution of NHE3 immunoreactivity out of the brush-border microvilli to intermicrovillar cleft and endosomal membranes ( 42 ); thesame conclusion was reached when analyzed by subcellular fractionation, NHE3 redistributed from lower-density membranes enriched in markers ofapical microvilli to higher-density membranes enriched in markers ofintermicrovillar cleft, dense apical tubules, and endosomes ( 42, 47 ). These same techniques were applied to the kidneys afteracute phenol injection to test the hypothesis that NHE3 wouldredistribute into the brush border in response to the increased sympathetic efferent activity; the alternative hypothesis was that theNHE3 would retract out of the brush border in response to thehypertension per se, identical to the response to arterial constriction. Representative immunoblots of NHE3 in 12 gradient fractions from saline- and phenol-injected kidneys are shown in Fig. 2 A. Samples were pooled intothree windows to simplify analyses, as described in METHODS. After phenol injection, a significant fraction ofNHE3 (expressed as percentage of total in the gradient) shifts out ofWIII into WI (Fig. 2 B ): NHE3 in WI increased from 13.07 ± 1.97 to 27.15 ± 4.08% of total, NHE3 in WII remainedunchanged (63.7 ± 3.53 to 61.45 ± 2.15%), and NHE3 in WIIIdecreased from 22.79 ± 4.75 to 10.79 ± 2.01% after phenolinjury ( P saline, assessed by ANOVA andfollowed by paired Student's t -test). These results supportthe hypothesis that phenol injury provokes a redistribution of NHE3from intracellular and intermicrovillar membrane pools to the apicalmicrovilli, a response that could favor increased salt and watertransport and the generation and persistence of hypertension afterphenol injury.

Fig. 2. Redistribution of Na /H exchanger type 3 (NHE3) to low-density membranes in response to phenolinjection and its inhibition by prior denervation. Renal cortex fromsaline- and phenol-injected animals were removed at 50-60 min andsubjected to subcellular fractionation on sorbitol density gradientscollected as 12 fractions. A : typical immunoblots of NHE3 ina constant volume of each fraction from saline- vs. phenol-injectedrenal cortices are shown. On the basis of previous analyses( 42 ), fractions were pooled into three windows: Window I( fractions 3-5 ) is enriched in apicalbrush-border markers; Window II ( fractions 6-8 )contains mixed membranes with markers from apical, intermicrovillarcleft, and dense apical tubule markers; and Window III( fractions 9-11 ) is enriched in endosomal andlysosomal markers. B : summary of NHE3 distribution in threewindows, expressed as the percentage of the total signal in all threewindows. Values are means ± SE; n = 6 in eachgroup. * P t -test. C : resultsof 2 sets of experiments in which the left kidneys were denervated 30 min before phenol injection. Immunoblots of a constant volume of eachfraction from rats with renal denervation alone or denervation followedby phenol injection are shown.
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NHE3 distribution in denervation vs. denervation followed by phenolinjection. Renal injury activates renal afferent pathways, increasesnorepinephrine release from the posterior hypothalamus, activates renalefferent pathways, and raises blood pressure. To test the hypothesisthat the activation of renal efferent pathways provokes the apicalredistribution of NHE3, we performed two sets of experiments in whichthe left kidney was denervated before phenol injection. As previouslyshown by Ye et al. ( 44 ), we found thatdenervation of the left renal nerves before the phenol injection intothe left kidney prevented the increase in blood pressure (not shown). As shown in Fig. 2, denervation per se did not affect the distribution pattern of NHE3 compared with the saline-injected kidney; however, denervation before phenol injection did prevent the redistribution ofNHE3 to WI (Fig. 2 C ). This provides direct evidence that the NHE3 redistribution to apical brush border in phenol injury (Fig. 2, A and B ) is associated with the activated renalefferent pathways and not to the local effects of the injected phenol." Q' f1 z- z3 H+ p# |# J5 a" Q# b5 K5 g

NHE3 distribution in phenol injury vs. arterialconstriction-induced hypertension. The change in the density gradient distribution pattern of NHE3 afterphenol injection-induced hypertension is reciprocal to that seen withacute hypertension due to arterial constriction, providing evidence forbidirectional regulation of NHE3 between apical microvilli andintermicrovillar/subapical membranes. In our previous study of thearterial constriction model of acute hypertension, blood pressure wasraised 50-70 mmHg ( 42, 47 ), whereas after phenolinjection blood pressure increased 20-30 mmHg, which allows forthe possibility that the distinct responses were a function of thedifferent levels of hypertension. NHE3 redistribution responses werereexamined by immunocytochemistry after blood pressure was increased by20-30 mmHg in both the phenol-induced and thearterial-constriction models of hypertension. Thirty minutes aftersaline or phenol injection or arterial constriction, kidneys were fixedin situ for another 20 min, as described in METHODS. Doublelabeling was performed on cryosections harvested from each of the threegroups. NHE3 was imaged with polyclonal NHE3-C00 with FITC-conjugatedanti-rabbit secondary, and villin, the actin bundling protein localizedto the microvilli, was imaged with monoclonal anti-villin with Alexa568-conjugated anti-mouse secondary antibody. We previouslydemonstrated that the subcellular distribution of villin was unalteredduring acute hypertension, so it provides a consistent backgroundmarker for the microvilli as the NHE3 redistributes ( 47 ).In saline-injected rats, the staining of NHE3 is restricted to thebrush border, as evidenced by colocalization with staining for villin(Fig. 3, top ). When bloodpressure is increased 20-30 mmHg by arterial constrictionhypertension (Fig. 3, bottom ), NHE3 moves out of the apicalbrush-border microvilli, leaving the tops of the villi stained red withanti-villin, NHE3 is detected in the intermicrovillar cleft regionwhere it coincides with villin (Fig. 3, yellow, arrow), and NHE3appears in subapical vesicles where it does not overlay villin (Fig. 3,green, arrowhead). This response is indistinguishable from thatobserved when blood pressure is increased 50-60 mmHg by arterialconstriction. After phenol injection associated with a 20-30 mmHghypertension, NHE3 remained colocalized with villin staining (Fig. 3, middle ). This technique was not sensitive enough to detectthe low levels of subapical NHE3 at baseline blood pressure, thus noredistribution was evident, but the finding provides strong visualconfirmation that NHE3 was not internalized during phenolinjury-induced hypertension. This comparison demonstrates that althoughblood pressure was increased 20-30 mmHg over baseline in bothanimal models, NHE3 is not internalized from apical membranes duringphenol injury-induced hypertension as it is during arterialconstriction hypertension.

Fig. 3. Effect of acute hypertension induced by 50 µl 10% phenolinjection vs. arterial constriction on NHE3 subcellular distribution.NHE3 was detected in renal proximal tubules from rats with salineinjection ( top ), phenol injection with an accompanying 20- to 30-mmHg increase in blood pressure ( middle ), or arterialconstriction with an accompanying 20- to 30-mmHg increase in bloodpressure ( bottom ). Kidneys were fixed in situ with 2%paraformaldehyde, 75 mM lysine, and 10 mM Na-periodate, pH 7.4, duringthe last 20 min. Sections were double labeled with polyclonalanti-rabbit NHE3-C00 antibody and then FITC-conjugated anti-rabbitsecondary antibody, and with monoclonal anti-villin antibody and thenAlexa 568-conjugated anti-mouse secondary antibody. NHE3 staining isgreen (arrowhead), villin staining is red, and overlapping NHE3 andvillin appears yellow (arrow). Bar = 10 µm.

Effect of acute phenol injury on the distributions of other apicalmembrane proteins. To determine the specificity of the redistribution of NHE3 during acutephenol injury, the distribution of NaPi2 was investigated. NaPi2 ismainly expressed in the proximal tubule apical brush border. NaPi2 hasbeen shown to move from apical membranes to intracellular membranes inresponse to acute hypertension due to arterial constriction and duringa high-P i diet ( 24, 47 ). Figure 4 A shows representativeimmunoblots of NaPi2 and summarized data expressed as percentage oftotal in the three windows from saline- vs. phenol-injected rats. Inphenol-injected rats, NaPi2 increased in WI from 9.5 ± 1.62 to18.7 ± 1.45% of total, NaPi2 decreased in WII from 68.72 ± 1.95 to 59.76 ± 2.21% of total, and there was no change in WIII( P by ANOVA and followedby paired Student's t -test). The results indicate thatNaPi2 may, similarly to NHE3, move from intermicrovillar cleft regionand/or dense apical tubules (WII) to apical membranes (WI) duringphenol injury-induced hypertension.+ V8 N9 a' ]1 q  r, D2 c' D3 U$ Q% M

Fig. 4. Na -P i cotransporter 2 (NaPi2)and dipeptidyl-peptidase IV (DPPIV) redistribute to low-densitymembranes in response to 50 µl 10% phenol injection. A :typical immunoblots of NaPi2 in a constant volume of each fraction fromsaline- vs. phenol-injected renal cortices and summary of NaPi2distribution in three windows, expressed as the percentage of the totalsignal in all three windows. B : typical immunoblots of DPPIVin a constant volume of each fraction from saline- vs. phenol-injectedrenal cortices and summary of DPPIV distribution in three windows,expressed as the percentage of the total signal in all three windows.Values are means ± SE, n = 6 in each group.* P t -test.

The distribution of two additional classic apical membrane proteinsalso changed in a manner similar to that of NHE3 and NaPi2 after phenolinjury. DPPIV, detected by immunoblot at 105 kDa, increased in WI from19.21 ± 1.59 to 28.58 ± 2.36% of total during phenolinjury (Fig. 4 B ). Alkaline phosphatase activity (percentage of total) increased in WI from 15.96 ± 0.87 to 28.6 ± 2.36%, whereas activity in WII decreased from 60.47 ± 1.76 to52.73 ± 1.73% and WIII decreased from 19.89 ± 1.5 to15.14 ± 1.3%, evidence for redistribution from intracellular andintermicrovillar cleft regions to the brush border along with NHE3 andNaPi2 (Fig. 5 A ). For both theDPPIV and the alkaline phosphatase shifts, P as assessed by ANOVA and followed by paired Student's t -test.9 H4 z" u( r2 e

Fig. 5. Alkaline phosphatase andNa -K -ATPase activity distributions in phenolinjury-induced hypertension. A : alkaline phosphatase,expressed as percentage of total activity in all three windows,redistributes to low-density membranes in response to 50 µl 10%phenol injection. B : Na -K -ATPaseactivity, assessed as K -dependent p -nitrophenylphosphatase did not change in phenol injection-induced hypertension.Values are means ± SE; n = 7 in each group.* P t -test.1 ?1 d' ^) j2 z8 I

Effect of acute phenol injury on basolateral membraneNa -K -ATPase. We previously determined that renal corticalNa -K -ATPase was inhibited in response toarterial constriction hypertension ( 26 ), so weinvestigated whether Na -K -ATPase activity wasaltered during phenol injury-induced hypertension. The subcellulardistribution of Na -K -ATPase activity in renalcortex (Fig. 5 B ) indicates a peak of Na -K -ATPase activity in WI that did notchange in activity or distribution pattern after acute phenol injury.$ d' y- \2 ?+ ]% |6 R9 o/ _
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DISCUSSION
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Abundant evidence has accumulated supporting a role of increasedSNS activity in renal injury-induced hypertension. Campese andcolleagues ( 10, 43, 44 ) developed a rat model of renal injury-induced hypertension caused by an injection of 50 µl 10% phenol to the lower pole of one kidney, which leads to an immediate elevation of norepinephrine secretion from posterior hypothalamus and arise in blood pressure. They also measured an increase in plasmanorepinephrine level and renal sympathetic activity recorded directlyfrom renal nerves after phenol injection. The injury per se is notsufficient to provoke the hypertension, because renal denervationbefore phenol injection prevents the increase in blood pressure andnorepinephrine secretion from posterior hypothalamus ( 44 ).Recent results from the same laboratory indicate that afferent impulsestriggered by the phenol injury activate ANG II formation in brainnuclei, which inhibits IL-1 and nNOS, leading to activation ofcentral and peripheral SNS activity. Injection of the ANG IIAT 1 receptor antagonist losartan (into the lateralventricle or intravenously) inhibited the effect of phenol on bloodpressure and increased IL-1 and nNOS mRNA in three regions of thebrain ( 45 ). In addition, the -adrenergic receptorblocker phentholamine normalized blood pressure (45a), allsupporting a role of -adrenergic receptor activation in the genesisof hypertension in this model. The effect of phenol injury onblood pressure is long lasting; 5 wk after phenol injection, the siteof injection is reduced to a microscopic scar while hypertension persists ( 43 ). The phenol-injected kidney is a necessarycomponent of the chronic hypertension because its removal at 4 wknormalizes blood pressure, likely because of elimination of the renalafferent impulses ( 10 ). Taken together, these studiessuggest that renal and central SNS activation are responsible forphenol injury-induced hypertension. Our present study extends theseobservations to the level of molecular mechanisms regulating proximaltubule Na transporters in this neurogenic hypertensivemodel. Acute phenol injection provoked a rapid redistribution of NHE3and NaPi2 to the apical microvilli, a response that may contribute tothe generation or maintenance of hypertension in this model.4 g+ \: v3 G( R$ I7 x  A

Formal analysis of the molecular mechanisms of SNS activation ofNa transport in vivo have not been previously conducted orreported; however, there are a number of studies on the in vitroeffects of norepinephrine on Na /H exchangers.The proximal tubule contains numerous -adrenergic receptor bindingsites ( 19, 37, 38 ) and conditions that increase receptornumber or postreceptor components responsible for 1 - and -adrenergic-mediated Na reabsorption in proximal tubulecan contribute to Na retention and elevated blood pressure( 19 ). Liu and colleagues ( 22, 23 ) found thatproximal nephron Na /H exchange transportactivity is increased by activation of 1A - and 1B -adrenergic receptor subtypes facilitated by the MAPKsignaling pathway. The findings of the present study provide the firstdirect in vivo evidence that SNS stimulation activates apicalNa /H exchange activity by increasing NHE3transporters at the apical surface, a response that can be prevented byrenal denervation. The increase in apical NHE3 may be accomplished bydecreasing endocytosis or increasing exocytosis from intracellularstores, or both. Because the subapical endosomal pool of NHE3 isdifficult to detect at baseline blood pressures by confocal microscopy, it is quite plausible that the apical NHE3 and NaPi2 accumulate due todepressed endocytosis.

In vivo studies have provided mixed results regarding adrenergicregulation of basolateral Na -K - ATPase inisolated and cultured renal proximal tubules. Norepinephrine was foundto increase solute and fluid reabsorption andNa -K -ATPase activity ( 1, 3 ) inisolated proximal tubules, an effect that may be driven by increasedapical Na entry ( 34 ). In another system,adrenergic stimulation drives exocytic insertion of Na pumps into the plasma membrane of cultured lung cells ( 4 ). However, Holtback et al. ( 18 ) concluded thatnorepinephrine has no net effect on proximal tubuleNa -K -ATPase activity because of combinedactivation of - and -adrenergic receptors in the proximal tubule.In the present study, there was no effect of phenol injection-inducedhypertension on Na -K -ATPase activity inisolated membranes resolved on sorbitol gradients. There was a slighttendency to redistribute Na -K -ATPase from WIIto the WI basolateral membrane peak, which may reflect insertion ofNa -K -ATPase from intracellular vesicles tothe plasma membranes, similar to the effect of adrenergic agents incultured lung cells ( 4 ), but establishing this willrequire an improved fractionation strategy or pharmacologicalmanipulation of - vs. -adrenergic receptor levels. In contrast,our laboratory previously determined that an acute increase in bloodpressure by artery constriction inhibits proximal tubuleNa -K -ATPase activity measured in isolatedmembranes ( 25, 47 ). It is possible that the opposingforces of SNS stimulation to increase activity and hypertension todecrease activity may counteract each other, an issue that could betested by SNS stimulation in a setting of servocontrolled bloodpressure. Can Na reabsorption increase without a change inNa -K -ATPase activity?Na -K -ATPase activity in vivo in the tubule isvery high to start with ( 28 ) and may indeed be activatedby increased Na availability or adrenergic stimulationwhile not detected enzymatically in a V max assayin broken membranes in vitro.3 Y$ H  K/ k! G' O
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Regarding the phenol injury itself, the hypertension occurs in the faceof minimal renal injury (1- to 2-mm wide) ( 43 ). In thisstudy, before cell fractionation, a small area surrounding theinjection site was removed, and areas beyond this injection areaappeared normal when examined by electron microscopy ( 43 ). Activation of the SNS reflex does not appear to be the result of anunspecific renal injury as lesions of the same dimensions caused byburning, administration of alkali (NaOH), acid (HCl), or methanol donot raise blood pressure (not shown). In addition, phenol injections toother sites, including the spleen and peritoneum, change blood pressureonly transiently over 44 ).- R- F/ [2 i8 d! m5 ]- c

We have previously reported that an acute increase in arterial bloodpressure brought about by arterial constriction provokes a rapiddecrease in proximal tubule Na reabsorption, the key toincreasing NaCl at the macula densa, which activates tubuloglomerularfeedback to autoregulate RBF and GFR ( 13, 14, 47 ). Theaccompanying natriuresis and diuresis are a compensatory response torestore elevated blood pressure toward normal levels ( 16 ).Using C Li as a measure of volume flow out of the proximaltubule, we have consistently observed that arterial constrictionhypertension causes a three- to fourfold increase in C Li and urine output ( 42, 47 ). The role of proximal tubuleNa reabsorption, estimated by C Li, in thegeneration or maintenance of hypertension has been assessed bydifferent investigators with differing results ( 12 ). Thisis likely because elevated Na and volume reabsorption maybe causal to some varieties of hypertension, whereas decreasedNa and volume reabsorption may be compensatory in othervarieties of hypertension. An increase in proximal reabsorption hasbeen demonstrated in unanesthetized spontaneously hypertensive rats ( 7 ) and in hypertensive patients ( 11, 27, 35 ). A decrease in proximal Na transport inhypertensive patients ( 32, 41 ) or no change ( 17, 36 ) has been reported as well. In the present study, hypertension caused by acute phenol injury did not cause a significant change in either urinary output or C Li, which could beexplained by the combined effect of SNS activation to increaseNa and volume reabsorption and elevated blood pressure,which would evoke a compensatory decrease in Na and volumereabsorption. Whether there is a significant increase in proximaltubule volume reabsorption associated with the Na transporter redistribution during phenol-induced hypertension is animportant question that remains to be answered. In any case, thehypertension evoked by the acute phenol injection does not lead to thehomeostatic compensation known as "pressure natriuresis" or to thethree- to fourfold increase in C Li observed during arterial constriction hypertension, evidence for a significant resetting of therenal function curve ( 16 ).' [. j! _1 M) j0 A9 J& ?& V  E2 B
$ _: R$ B# m2 C! b- w* @( ?
The distinct responses to hypertension provoked by phenol injection vs.arterial constriction persist at the molecular level. Our laboratorypreviously studied the molecular mechanisms responsible for thedecrease in proximal tubule Na reabsorption duringarterial constriction hypertension and discovered there is anaccompanying retraction of transport competent NHE3 as well as NaPi2from apical brush border to intermicrovillar cleft and subapicalmembrane pools and inhibition of basolateral Na -K -ATPase ( 25, 42, 47 ). Incontrast, during acute phenol injury-induced hypertension, NHE3 andNaPi2 redistribute in the opposite direction from subapical endosomesto the apical brush border. The two hypertension-dependent patternsappear remarkably distinct when analyzed by confocal microscopy.Although blood pressure was raised to the same extent (~20-30mmHg) in both models, proximal tubule NHE3 was internalized in thearterial constriction hypertension, presumably a compensatory response,but not in the phenol injection-induced hypertension. By confocalanalysis, there is no obvious shift of NHE3 from subapical stores tothe microvilli. There are a variety of interpretations that could beresolved by electron microscopy analysis; NHE3 may redistributelaterally from stores in the intermicrovillar cleft to the microvilli(decreased traffic to the cleft or increased traffic to the microvilli)or redistribution from the endosomal pools (decreased internalizationor increased exocytosis) is below the level of detection byimmunofluorescence, because the concentration of NHE3 is too low atbaseline. Perhaps SNS activation without hypertension wouldlead to a more obvious redistribution of NHE3 to the brush border.

Girardi et al. ( 15 ) have studied proteins thatassociate with proximal tubule NHE3 by coimmunoprecipitation anddiscovered that DPPIV associates with NHE3 predominantly in themicrovillar fraction in which NHE3 is active, as opposed to theintermicrovillar cleft region, suggesting that the association mayaffect NHE3 surface expression and/or activity. During phenolinjection-induced hypertension, we found that DPPIV was recruited tothe apical membranes in WI along with NHE3, which is consistent withthe findings of Girardi et al. ( 15 ) that thiswould indicate increased association and NHE3 activity in themicrovilli. In contrast, during arterial-constriction hypertension,DPPIV is internalized along with NHE3 ( 47 ), which is alsoconsistent with a functional link between NHE3 and DPPIV.8 q9 r# e" B/ Z! {+ [8 [# T
( T9 x& }; k6 y/ W3 i/ l
The pattern of redistribution of NaPi2 to WI apical-enriched membranesduring phenol injury-induced hypertension was distinct from that ofNHE3 redistribution to WI; NHE3 was redistributed from WIII(intermicrovillar cleft, dense apical tubules, and endosomes) and NaPi2was redistributed from WII (apical, intermicrovillar cleft, and denseapical tubule). This finding suggests that NaPi2 is translocated fromintermicrovillar cleft or dense apical tubules, not intracellularendosomes, to the apical surface. This interpretation isconsistent with Murer et al. ( 31 ), demonstrating thatwhen NaPi2 is translocated from apical brush border to endosomes during parathyroid hormone treatment or high dietary P i, it isdirectly routed to lysosomes for degradation, and recovery of transport activity requires de novo synthesis of NaPi2 rather than redistribution from an endosomal pool. A relevant in vivo study demonstrated that a rapid adaptive increase in renal proximal tubule apical NaPi2abundance in response to acute administration of a low-P i diet is independent of de novo protein synthesis mediated bymicrotubule-dependent translocation of presynthesized NaPi2 protein tothe apical brush border ( 21, 24 ), suggesting the existenceof an intracellular NaPi2 pool of limited size that could be involvedin the fine adjustment of renal P i reabsorption. Whetherthe NaPi2 that moved to WI is nascent NaPi2 en route to the apicalmembrane or from a recruitable pool was not determined.
5 B8 e, A- Z* T- Y3 f
The activity of the apical brush-border marker alkaline phosphatase isalso differentially regulated by hypertension induced by phenol injuryvs. arterial constriction. After phenol injection, alkaline phosphataseactivity shifts to the apical membrane-enriched WI from WII and WIII.During arterial constriction, total alkaline phosphatase activity isdecreased and the peak shifts out of lower density apical membranes( fractions 3-5, WI) into higher density membranes ( fraction 6, WII) ( 47 ). Theseresults suggest alkaline phosphatase may also be involved in theregulation of NHE3 and/or NaPi2 traffic through its association ordissociation with these apical transporters.# q0 }" W% T' K! z/ g
1 C. w+ ^) U% [4 P
In summary, in this neurogenic hypertensive model, acute phenolinjection provokes a rapid redistribution of NHE3 and NaPi2 to theapical microvilli. Renal denervation prevented the NHE3 redistribution,suggesting that SNS activation of proximal tubule Na transport in vivo is mediated by recruiting transporters to the apicalbrush border. Renal denervation also prevented the development ofhypertension, suggesting that the proximal tubule response may play arole in the generation or maintenance of the phenol injury-inducedhypertension. Finally, the results provide evidence for bidirectionalregulation of NHE3 and NaPi2 during hypertension; transporters may beinternalized consistently with a compensatory response, as observed inarterial constriction hypertension, or may be recruited to themicrovilli in a fashion that would contribute to hypertension, asobserved in neurogenic phenol injury-induced hypertension.3 H6 M+ _. p( Q' T" Y2 f

ACKNOWLEDGEMENTS

We are grateful to Michaela Mac Veigh for assistance with confocal microscopy., G: m1 ~9 {; v0 h
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