Chronic renal injury-induced hypertension alters renal NHE3distribution and abun

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作者：Li E.Yang, HuiqinZhong, Patrick K. K.Leong, AnjanaPerianayagam, Vito M.Campese,  Alicia A.McDonough作者单位：1 Department of Physiology and Biophysics and Division of Nephrology, Department of Medicine,University of Southern California Keck School of Medicine, Los Angeles,California 90089-9142 , O0 j6 i% v( W: V1 ]- F
                  
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          【摘要】
* O+ P5 p7 O7 }  U; t, A      Renal cortical phenol injection provokesacute sympathetic nervous system-dependent hypertension and a shift ofproximal tubule Na   /H   exchanger isoform 3 (NHE3) and Na   -P i cotransporter type 2 (NaPi2)to apical microvilli. This study aimed to determine whether proximaltubule (PT) Na   transporter redistribution persistschronically and whether the pool sizes of renal Na   transporters are altered. At 5 wk after a 50-µl 10% phenolinjection, blood pressure is elevated: 154 ± 8 vs. 113 ± 11 mmHg after saline injection. Cortical membranes were fractionated intothree "windows" enriched in apical brush border ( WI ),mixed apical and intermicrovillar cleft ( WII ), andintracellular membranes ( WIII ). NHE3 relative distributionin these windows, assessed by immunoblots and expressed as %total,remained shifted to apical from intracellular membranes ( WI :25.3 ± 3 in phenol vs.12.7 ± 3% in saline and WIII : 9.1 ± 1.3 in phenol vs. 18.9 ± 3% insaline). NaPi2 and dipeptidyl-peptidase IV also remained shifted to WI, and alkaline phosphatase activity increased 100.9 ± 29.7 ( WI ) and 51.4 ± 17.5% ( WII ) inphenol-injected membranes. Na   transporter total abundance[NHE3, NaPi2, thiazide-sensitive Na-Cl cotransporter,bumetanide-sensitive Na-K-2Cl cotransporter, Na-K-ATPase 1 - and 1 -subunits, and epithelialNa   channel (ENaC) - and -subunits] was profiled byimmunoblotting. Only cortical NHE3 abundance was altered, decreasing to0.56 ± 0.06. The results demonstrate that phenol injury provokesa persistant shift of PT NHE3 and NaPi2 to the apical microvilli, alongwith a 44% decrease in total NHE3, evidence for an escape mechanism that would counteract the redistribution of a larger fraction of NHE3to the apical surface by normalizing the total amount of NHE3 in apical membranes. 6 ~& @$ ^& ~1 o7 O# v; n# U/ W  y
          【关键词】 sodium transport membrane traffic sympathetic nervous system phenol
; F4 |; O2 R' a                  INTRODUCTION
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A MODEL OF NEUROGENIC HYPERTENSION provoked by intrarenal injection ofphenol into the cortex of a pole of one kidney was recently developedby Ye and Campese ( 8, 33, 34 ). In this model, a 50-µl10% phenol injection causes a rapid elevation of blood pressure,preceded by a rise in norepinephrine secretion from the posteriorhypothalamus and an increase in renal sympathetic nervous systemactivity. Renal denervation before phenol injection prevents thesympathetic nervous system activation as well as the rise in bloodpressure. These results are consistent with the interpretation thatthis phenol renal injury activates renal afferent pathways, increasesnorepinephrine release from the posterior hypothalamus, activates renalefferent pathways, and raises blood pressure. Interestingly, thehypertension becomes established and persists long after the site ofinjury recedes to the point when it is just a microscopic scar. Thecellular and molecular bases for the hypertension are not clearlyunderstood. One potential contributor is activation of sodium andvolume reabsorption mediated by renal efferent sympathetic nerve activity.$ n$ X7 @/ Z' Z( @$ F: g$ T7 J5 w
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A dynamic relationship between blood pressure and renal sodiumreabsorption is responsible, at least in part, for the blood pressureset point ( 15 ). Increases in sodium transport can be responsible for the generation and maintenance of hypertension, whereasdecreases in sodium transport may be evidence of homeostatic compensation for elevated blood pressure. For example, an experimental increase in blood pressure acutely decreases proximal tubule sodium reabsorption, which both increases NaCl delivery at the macula densa, atransglomerular feedback signal to normalize renal blood flow andglomerualr filtration rate, and causes pressure-natriuresis thatreduces extracellular volume, which in turn counteracts the hypertension ( 6, 9, 10 ). In contrast, if renal sodium reabsorption is elevated due to excess production of an antinatriuretic (e.g., aldosterone) ( 30 ) or to a mutated epithelial sodiumchannel (ENaC; Liddle's syndrome), then extracellular volume increases and blood pressure rises. The secondary hypertension depresses sodiumreabsorption at sites along the nephron, for example, the thiazide-sensitive Na   -Cl cotransporter (NCC)( 30 ), to match sodium excretion to sodium intake, apressure-natriuresis variant known as "escape." Although thesephenomena are well established as important for the maintenance ofextracellular volume and blood pressure, many questions remain regarding the molecular mechanisms responsible for regulation of sodiumtransporters along the nephron in compensating for hypertension or ingenerating and maintaining hypertension.- X+ q+ R; A$ J% w4 R- L5 s
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This laboratory has investigated the proximal tubule sodium transporterresponses during experimental acute hypertension induced by increasingperipheral resistance as well as in the spontaneously hypertensive rat(SHR). In both models, there was a retraction ofNa   /H   exchangers (NHE3) andNa   -P i cotransporters (NaPi) from the apicalbrush border to the intermicrovillar cleft and subapical endosomes, asdemonstrated by both subcellular fractionation and confocal microscopy( 21, 36, 37 ). In addition, there was a decrease inbasolateral Na-K-ATPase activity with the onset of hypertension in bothmodels ( 21, 37 ). Recently, we analyzed the acute response(30 min) to phenol injury in the rat cortex and discovered that NHE3and NaPi redistributed from intracellular membranes to the apical microvilli, mediated by sympathetic nervous system activation, aresponse that could contribute to increased sodium/volume status ( 32 ). Motivated by these findings, we aimed to determinethe chronic (5 wk) effects of phenol renal injury on proximal tubule sodium transporter distribution, namely, whether the proximal tubulesodium transporters would maintain a redistribution to the apicalmembranes or whether the hypertension would drive a retraction ofproximal tubule sodium transporters from the apical microvilli as seenin the increased peripheral resistance and SHR models. It has beenreported that the kidney can escape from certain sodium-retentiondisorders, such as hyperaldosterone states, by downregulating renalsodium transporters, such as the NaCl transporter of the distal tubule,to counteract the sodium retention ( 30 ). Therefore, in thepresent study we looked for evidence of escape during chronic phenolinjury-induced hypertension by examining the total pool size of renalsodium transporters along the nephron. The results demonstrate that theredistribution of NHE3 and NaPi2 to apical microvilli and Na-K-ATPaseactivity to the plasma membranes persists for 5 wk after phenol injuryand a decrease in cortical NHE3 abundance as evidence for a coincident escape mechanism in the same region of the nephron.0 g2 C& h0 z) s$ E6 _2 A
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EXPERIMENTAL PROCEDURES
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Animal preparation. Experiments were performed in male Sprague-Dawley (SD) rats(280-320 g body wt) that had free access to food and water. After anesthesia with an intramuscular injection of ketamine (Fort Dodge Laboratories) and xylazine (1:1, vol/vol, Miles), the left kidney wasexposed via a dorsal incision, 50 µl of 10% phenol or saline wereinjected into the lower pole of the renal cortex, the incision wassutured closed, and the rats were returned to the vivarium, where theyhad free access to food and water. After 5 wk, rats were anesthetizedas above and placed on a thermostatically controlled warming table tomaintain body temperature at 37°C. A polyethylene catheter was placedinto the carotid artery to record blood pressure. In one set, rats wereanesthetized with an intraperitoneal injection of pentobarbital sodium(35 mg/kg).
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Homogenization and subcellular fractionation. The procedure for subcellular fractionation of renal cortical membraneshas been described previously ( 38, 39 ). In brief, thenoninjected right kidney was cooled in situ by flushing with cold PBSand then excised. The renal cortices and medullas were dissected,homogenized in isolation buffer [5% sorbitol, 0.5 mM disodium EDTA,0.2 mM phenylmethylsulfonyl fluoride, 9 µg/ml aprotinin, and 5 mMhistidine-imidazole buffer (pH 7.5)] with a Tissuemizer (TekmarInstruments), and centrifuged at 2,000 g for 10 min; the pellet was rehomogenized and centrifuged, and the low-speedsupernatants (S o ) were pooled. The cortical S o was loaded between two hyperbolic sorbitol gradients and centrifuged at100,000 g for 5 h, and 12 fractions were then collectedfrom the top, diluted with isolation buffer, pelleted by centrifugation(250,000 g for 1.5 h), resuspended in 1 ml isolationbuffer, and stored at 80°C pending assay. To simplify themeasurements, fractions were pooled into three windows based onprevious analyses ( 31, 37 ): window I ( WI; fractions 3-5 ) is enriched in apicalbrush border and basolateral membrane markers, window II ( WII; fractions 6-8 ) is enriched inintermicrovillar cleft and dense apical tubule markers, and window III ( WIII; fractions 9-11 )is enriched in endosomal markers." O8 p, y* |1 H* P9 r+ D
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Immunoblot analysis and antibodies. To determine the distribution of proteins in the sorbitol gradient, aconstant volume of each window was assayed and expressed as thepercentage of the total in all three windows. To assess the total poolsize of transporters expressed along the nephron, a constant amount ofS o protein (2,000- g supernatant of homogenate) was analyzed. In all assays, one-half the volume or one-half the protein was also assayed to verify linearity of the detection system.Samples were denatured in SDS-PAGE sample buffer for 30 min at 37°C,resolved on 7.5% SDS polyacrylmide gels according to Laemmli( 20 ), and transferred to polyvinylidene difluoride membranes (Millipore Immobilon-P). Polyclonal antisera to NHE3 [NHE3-C00; McDonough laboratory ( 31 )] and to NaPi2 [J.Biber and H. Murer, University of Zürich, Zurich, Switzerland]were used at 1:2,000 dilution. Polyclonal antisera to dipeptidylpeptidase IV (DPPIV; M. Farquhar, Univ. of California at San Diego)were used at 1:1,000 dilution. A monoclonal antibody specific forNa-K-ATPase -subunit (464.6) (M. Kashgarian, Yale Univ.) was used at1:200 dilution. Polyclonal anti-Na-K-ATPase -subunit (McDonoughlaboratory) and a polyclonal antiserum to NaCl transporter (TSC; D. Ellison, Oregon Health and Science Univ.) were used at 1:500 dilution. Monoclonal anti-Na-K-2Cl transporter antibody (T4; C. Lytle, Univ. ofCalifornia at Riverside) and polyclonal anti-NHERF1 antibody (R-1046;E. Weinman, Univ. of Maryland School of Medicine) were used at 1:3,000dilution. Polyclonal antisera to ENaC - and -subunits (Chemicon) were used at 1:1,000 dilution. Except for - and -ENaC, all blots were incubated with Alexa 680-labeled goat anti-rabbit (Molecular Probes, Eugene, OR) or goat anti-rabbit IRDye800 or goatanti-mouse IRDye800 secondary antibody (both from LI-COR, Lincoln, NE),detected with an Odyssey Infrared Imaging System (LI-COR), andquantitated using the accompanying LI-COR software. - and -ENaCwere detected with the enhanced chemiluminescence (ECL) kit (AmershamPharmacia Biotech), and autoradiographic signals were quantified with aBio-Rad imaging densitometer with Molecular Analyst software. Multipleexposures of autoradiograms were analyzed to ensure that signals werewithin the linear range of the film.3 m: V, T/ O2 ^/ f$ }& h
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Indirect immunofluorescence. Immunofluorescence analysis was conducted as described in detailpreviously ( 31 ). In brief, the kidney contralateral to thesaline or phenol injection was fixed in situ (without perfusion offixative to avoid changing renal perfusion pressure), cut in half on amidsagittal plane, and postfixed in periodate-lysine-paraformaldehyde, incubated overnight in 30% sucrose in PBS, embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA),and frozen in liquid nitrogen for 5-µm cryosectioning. Sections wereincubated with 1% SDS in PBS for 4 min for antigen retrieval ( 7 ), SDS was removed by washing in PBS, and then sectionswere blocked with 1% bovine serum albumin in PBS. Double labeling was performed by incubating with polyclonal antiserum NHE3-C00 and amonoclonal antibody against villin (Immunotech, Chicago, IL) and thendetected with a mixture of FITC-conjugated goat anti-rabbit (CappelResearch Products, Durham, NC) and Alexa 568-conjugated goat anti-mouse(Molecular Probes), as described previously ( 31 ). Slideswere viewed with a Nikon PCM Quantitative Measuring High-Performance Confocal System equipped with filters for both FITC and TRITC fluorescence attached to a Nikon TE300 Quantum upright microscope. Images were acquired with Simple PCI C-Imaging Hardware andQuantitative Measuring Software and processed with Adobe PhotoDeluxe(Adobe Systems, Mountain View, CA).5 O! t  F; u$ L3 h: M
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Assays. Na   -K   -ATPase activity was measured bythe potassium-dependent p -nitrophenyl phosphatase(K   - p NPPase) reaction ( 27 ),alkaline phosphatase activity was measured as described( 25 ), and protein concentration was measured with the BCAassay kit (Pierce Technology, Iselin, NJ).
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Quantitation and statistical analysis. Data are expressed as means ± SE. Differences were regardedsignificant at P ANOVA was applied to determine whether there was asignificant effect of treatment on the overall pattern. If significancewas established, the location of the difference in the pattern was assessed by two-tailed Student's t -test for paired samples.Differences in total cell sodium transporters were assessed bytwo-tailed Student's t -test for paired samples.. `) I3 n3 C, `! E& m* p+ y8 t

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Effects of intrarenal phenol injection on systolic arterialpressure. Ye et al. ( 33, 34 ) have reported that a limited renalinjury through an intrarenal injection of 50 µl of 10% phenol caused an immediate and permanent form of neurogenic hypertension. In thisstudy, we independently verified this finding in a different laboratory. Five weeks after 50 µl of 10% phenol or saline were adminstered in the lower pole of one renal cortex, arterial blood pressure was measured in ketamine- and xylazine-anesthetized rats ( n = 6) via arterial cannulation, and systolic bloodpressure was significantly elevated in the phenol-injected rats(129 ± 3 mmHg) compared with the saline-injected rats (115 ± 3 mmHg). In a subgroup of the ketamine/xylazine-anesthetized rats( n = 3), blood pressure was measured by tail cuffbefore injection and 5 wk thereafter in conscious rats: phenolinjection increased systolic pressure from 123 ± 1 to 157 ± 2 mmHg, whereas injection of 50 µl saline did not change bloodpressure. Because we and others have previously reported that bloodpressure measured in anesthetized SHRs is higher with pentobarbitalsodium compared with ketamine/xylazine ( 22, 37 ), we alsochecked systolic arterial pressure in pentobarbital sodium-anesthetizedrats ( n = 3) 5 wk after phenol injection (rats not usedin further experiments) and found that the measured blood pressure wasindeed higher than that in the ketamine/xylazine group (154 ± 8 vs. 113 ± 11 mmHg in phenol-injected vs. saline-injected rats).This may be attributed to the observation that xylazine, a centrallyacting 2 -adrenergic agonist, promotes urinary sodiumexcretion by a renal nerve-dependent pathway ( 24 ). Thesemeasurements confirm the previous report that a single phenol injectionprovokes persistent hypertension ( 33 ) and that themeasured blood pressure is higher with pentobarbital sodium anesthesiacompared with ketamine/xylazine.
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Immunoblot detection of NHE3 distribution in phenol injury-inducedchronic hypertension. NHE is the major route for apical sodium entry across the proximaltubule, and NHE3 is responsible for virtually all theNa   /H   exchange activity in this region( 1, 4 ). We recently discovered that the acute hypertensionestablished 30 min after intrarenal injection of 50 µl of 10% phenolis associated with redistribution of NHE3 immunoreactivity fromintermediate-density membranes enriched in markers of intermicrovillarcleft and endosomal pools to lower density membranes enriched in apicalbrush-border microvillar markers, a response that may contribute to thegeneration of phenol injury-induced hypertension and a response blockedby prior renal denervation ( 32 ). Our previous studies in amodel of chronic hypertension found that as chronic hypertensiondeveloped with age in the SHR, NHE3 redistributed in the oppositedirection: from lower density membranes enriched in markers of apicalmicrovilli to higher density membranes enriched in markers of theintermicrovillar cleft and endosomes, a response also verified byconfocal microscopy ( 36 ), which provides evidence for ahomeostatic compensation to the developing hypertension( 21 ). These disparate findings stimulated us to examinewhether NHE3 would remain shifted to the apical membranes, as evidencedin the acute response to phenol injury, or would retract to theintermicrovillar membranes, as evidenced in the chronic hypertensiveSHR. NHE3 distribution was studied in the contralateral kidney 5 wkafter phenol or saline injection. Figure 1 A shows representativeimmunoblots of NHE3 in the renal cortical membranes fractionated intothe three defined windows ( WI is enriched in apicalbrush-border markers alkaline phosphatase, DPPIV, and NHE3; WII contains most of the intermicrovillar cleft markermegalin as well as the apical markers; and WIII is enrichedin megalin as well as the endosomal marker rab 5a and the lysosomalmarker -hexosaminidase) ( 31, 37 ). Because a constantvolume, rather than protein, of each window was analyzed, the totalimmunoreactivity in the saline-vs.-phenol samples is not expected to beidentical. The differences in total NHE3 are analyzed subsequently. Theresults indicate that the phenol injection-induced shift of NHE3 out of WIII into WI seen at 30 min persists for 5 wk(Fig. 1 B ): WI NHE3, expressed as %total NHE3 inthe gradient, contains 25.3 ± 3% in the phenol group and12.7 ± 2.7% in the saline group; WII NHE3 isunchanged, 65.6 ± 2.3% in the phenol group and 68.4 ± 1.9% in the saline group; WIII NHE3 contains 9.1 ± 1.4% in the phenol group and 18.9 ± 3.4% in the saline group. These results indicate that there is a persistent signal for net traffic of sodium transporters to the surface in the chronichypertensive phenol-injected group compared with saline-injectedcontrols. The pattern is distinct from the internalization of NHE3 that was observed in the chronic hypertension of SHRs or Goldblatt twokidney, one-clip (2K1C) hypertension ( 21, 36 ).
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Fig. 1. Na   /H   exchanger isoform 3 (NHE3)redistributes to low-density membranes during phenol injury-inducedchronic hypertension. Five weeks after injection of 50 µl of 10%phenol or 50 µl saline into the lower pole of the left kidney, ratswere killed and renal cortices from the right kidneys were removed andsubjected to subcellular fractionation in sorbitol density gradientscollected as 12 fractions. Based on previous analyses ( 31, 37 ), fractions were pooled into 3 windows: window I ( fractions 3-5 ), enriched in apicalbrush-border markers; window II ( fractions6-8 ), enriched in intermicrovillar cleft markers; and window III ( fractions 9-11 ),enriched in endosomal markers. A : typical immunoblots ofNHE3 from saline- vs. phenol-injected rats. Volumes assayed wereadjusted to ensure that signals were within the linear range ofdetection, and 1/2 the volume was loaded to validatequantitation in the linear range. B : summary of NHE3distribution in the 3 windows. From the autoradiograms, density/µlwas calculated and corrected for the total volume per window, and NHE3distribution is expressed as the percentage of the total density signalin all 3 windows. Values are means ± SE; n = 5/group. * P t -test.! C, [+ e1 U; k
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Indirect immunocytochemistry of NHE3 in phenol injury-inducedchronic hypertension. NHE3 distribution in phenol-induced hypertension was also visualized byimmunocytochemistry. Five weeks after phenol or saline injection,kidneys were fixed in situ for 20 min as described in EXPERIMENTAL PROCEDURES. Cryosections harvested from bothgroups were double labeled: NHE3 was detected with polyclonal NHE3-C00- and FITC-conjugated goat anti-rabbit secondary antibody; villin, theactin-bundling protein found in the microvilli, was detected withmonoclonal anti-villin together with Alexa 568-conjugated goatanti-mouse secondary antibody. In saline-injected rats, the staining ofNHE3 is restricted to the brush border, as evidenced by colocalizationwith staining of villin (Fig. 2, top ). In phenol injury-inducedhypertension, there was no discernable change in NHE3 distributionpattern, as it still colocalized with villin staining (Fig. 2, bottom ). While not quantitated directly, there is evidencefor lower levels of NHE3 in the phenol injury group, a point confirmedby immunoblots and discussed in Fig. 7. This technique provides visualconfirmation that NHE3 is not distributed out of the villi and isinternalized to endosomes during phenol injury-induced chronichypertension as reported in the chronic hypertension in both SHRs andGoldblatt 2K1C ( 36 ) and in acute hypertension byincreasing peripheral resistance ( 31, 36 ).# @: M) C4 \  M

. a0 A  T$ k; ?# K! x) WFig. 2. Confocal microscopy verified no evidence for NHE3internalization during phenol injury-induced chronic hypertension. NHE3was detected in renal cortex containing proximal tubules of kidneyscontralateral to the site of saline injection ( top ) orphenol injection ( bottom ). Kidneys were fixed in situ withperiodate-lysine-paraformaldehyde for 20 min. Sections weredouble-labeled with polyclonal anti-rabbit NHE3-C00 antibody and thenFITC-conjugated anti-rabbit secondary antibody and with monoclonalanti-villin antibody and then Alexa 568-conjugated anti-mousesecondary. NHE3 staining is green, villin staining is red, andoverlapping NHE3 and villin appears yellow. Bar = 10 µm.9 o4 `  ~+ Q% o+ i( n' H/ Y5 b
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Distribution of other apical membrane proteins in phenolinjury-induced hypertension. In our previous study, we demonstrated that 30 min after acute phenolinjection the percentage of proximal tubule NaPi2 in WI apical microvilli increased from 9.5 ± 1.6 to 18.7 ± 1.5%and NaPi2 in WII decreased by a similar percentage. DPPIV,an NHE3-associated protein in microvilli ( 14 ), increasedin WI from 19.2 ± 1.6 to 28.6 ± 2.4% of thetotal 30 min after phenol injection. Figure 3 A summarizes the NaPi2distribution 5 wk after phenol injection during the chronichypertension phase. There was a significant difference in the NaPi2distribution in the phenol- compared with saline-injected rats,indicating a shift out of WII into WI : NaPi2 in WI, expressed as %total in the gradient, was 22 ± 2.3 (phenol) vs. 11.2 ± 2.1% (saline); WII NaPi2 was60.7 ± 1.8 (phenol) vs. 70.7 ± 2.4% (saline); and therewas no change in WIII. The results indicate that NaPi2 mayredistribute, as in the acute phase of hypertension (30 min afterphenol injection), from the intermicrovillar cleft region and/or denseapical tubules ( WII ) to apical membranes ( WI )during phenol injury-induced hypertension. The redistribution of theclassic apical membrane protein DPPIV also persisted, similar to thatof DPPIV at 30 min after phenol injection: a slight but significantincrease in WI to 24.4 ± 11% of total from 18.1 ± 8.1% 5 wk after phenol injury (Fig. 3 B ), supporting afunctional link between NHE3 and DPPIV.# X: F9 d9 F, q" \; k4 o4 u

" y* a8 R% i6 h! qFig. 3. Na   -P i cotransporter type 2 (NaPi2) and dipeptidyl peptidase IV (DPPIV) redistribute to low-densitymembranes during phenol injury-induced chronic hypertension. NaPi2 andDPPIV were prepared and assayed as described in Fig. 1. A :typical immunoblots of NaPi2 from saline- vs. phenol-injected rats andsummary of NaPi2 distribution in 3 windows expressed as the percentageof the total signal in all 3 windows. B : typical immunoblotsof DPPIV from saline- vs. phenol-injected rats and summary of DPPIVdistribution in 3 windows expressed as the percentage of the totalsignal in all 3 windows. Values are means ± SE; n = 5/group. * P t -test.2 Z" C3 j5 Y8 I6 {. g+ s$ J
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We previously reported that 30 min after phenol injection the activityof the classic apical microvillar marker alkaline phosphatase increased80% in the apical membranes in WI. Figure 4 shows alkaline phosphatase distributionand activity in the fractionated membranes from saline- andphenol-injected rats 5 wk after phenol injection. The results indicatea persistent and substantial activation of alkaline phosphataseactivity during the chronic phase of phenol injury-inducedhypertension: in WI, alkaline phosphatase activity was100.9 ± 29.7% higher in phenol- compared with saline-injected rats, and WII activity was 51.4 ± 17.5% higher (nochange in WIII activity).
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Fig. 4. Alkaline phosphatase distribution and activity in phenolinjury-induced chronic hypertension 5 wk after injection of 50 µl of10% phenol vs. 50 µl of saline. A : alkaline phosphataseactivity (µmolP i · h 1 · mg 1 )was increased in windows I and II. B :alkaline phosphatase distribution (%total) was unchanged. Values aremeans ± SE; n = 6/group. * P t -test.1 w( _( z; @* o3 g. d
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Basolateral membrane Na-K-ATPase activity in phenol injury-inducedhypertension. Our previous investigations suggest that renal cortical Na-K-ATPaseactivity falls as hypertension develops in both acute hypertension fromarterial constriction ( 22, 37 ) and chronic hypertension,as in the developing SHR ( 21 ). However, 30 min afterphenol injection we did not observe any change in Na-K-ATPase activityor distribution ( 32 ). During the chronichypertension phase 5 wk after phenol injection (Fig. 5 ), Na-K-ATPase activity wassignificantly shifted to the basolateral membranes found in WI, perhaps mechanistically similar to the report that thesympathetic -agonist isoproterenol increases surface expression ofNa-K-ATPase in cultured lung cells ( 2 ).' {" D% e* a5 [" ~4 c& K8 m
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Fig. 5. Na-K-ATPase activity and distribution in phenolinjury-induced chronic hypertension 5 wk after injection of 50 µl of10% phenol vs. 50 µl of saline. A : Na-K-ATPase activity,assessed as K   -dependent p -nitrophenylphosphatase (K   - p NPPase). B :Na-K-ATPase distribution (%total). C : total Na-K-ATPaseactivity in cortical and medullary membranes. Values are means ± SE; n = 6/group. * P t -test.
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: s# l" b9 z0 M. r! f/ \4 A* nProfiling of sodium transporter abundance in renal cortex andmedulla. To determine whether the total pool size of sodium transporters locatedalong the nephron was altered 5 wk after phenol injection duringchronic hypertension, S o samples (homogenates subjected toa 2,000- g spin to remove poorly homogenized bits) ofcortical and medullary membranes from saline- and phenol-injected rats were assayed and immunoreactivity was quantified. Figure 6 demonstrates the linearity of theinfrared imaging system (LI-COR). The relative distribution of thesetransporters in the cortex vs. medulla was also verified: NHE3, thethiazide-sensitive NCC, and NaPi2 were enriched in the cortex, thebumetanide-sensitive Na   -K   -2Cl cotransporter was expressed in the medulla, and Na-K-ATPase 1 - and -subunits were found in both the cortex andmedulla, with more in the latter. Figure 7 summarizes the immunoblots of these transporters and proteins in saline- vs. phenol-injected rats. Toensure linearity, each sample was run twice, with one-half the proteinloaded in the second lane. The densitometric quantitation is shown inTable 1. In the cortex, there was aremarkable decrease in NHE3 protein in phenol-induced hypertensive ratsto 58.1 ± 7.4% of that measured in the saline-injected controls.This fall in cortical NHE3 was region specific, as there was nosignificant change in NHE3 total abundance in the medulla. There wasalso a tendency toward a decrease in total renal cortical Na-K-ATPase activity (Fig. 5 C ), which parallels the decrease in apicalNHE3 abundance, but this did not reach statistical significance and wasnot paralleled by a change in total Na-K-ATPase subunit pool sizes(Table 1 ). There were no other significant differences inimmunoreactivity of the other sodium transporters or in the NHE3-associated proteins DPPIV and NHE regulatory factor (NHERF) inphenol-induced hypertension compared with saline-injected rats (Fig. 7,Table 1 ).* h7 }0 L1 i7 o- Y

2 D" h6 Q( |$ U2 Q5 W+ D" \Fig. 6. Detection of sodium transporter proteins in renalcortical and medullary membranes using LI-COR infrared imaging system.Sodium transporters were detected over a range of protein loadingamounts to define linear range for detection and quantitation. NHE3 at83 kDa, NaPi2 at 82 kDa, and thiazide-sensitiveNa   -Cl cotransporter (NCC) at 160 kDa areenriched in the cortex;Na   -K   -2Cl cotransporter (NKCC)at 160 kDa is detected in medulla; and Na-K-ATPase (NKA) 1 -subunit at 100 kDa and Na-K-ATPase 1 -subunit at 50 kDa are detected in both regions and arerelatively enriched in the medulla.  e: K7 @2 n5 ~) @3 y

5 Z( Y/ J3 p6 {- hFig. 7. Profiling of sodium transporter and associated proteinabundance in total membranes of renal cortex and medulla from phenolinjury-induced hypertension vs. saline-injected control rats. Samplesfrom individual rats were assayed. For every sample, 1/2 theprotein was run to ensure sample was in the linear range of detection.The amounts of protein loaded are 40/20 µg for NHE3,Na   /H   exchange regulatory factor (NHERF), andcortical -ENaC; 20/10 µg for NaPi2, NKCC, and medullaryNa-K-ATPase 1 -subunit; 15/7.5 µg for DPPIV; 32/16 µgfor NCC and cortical Na-K-ATPase 1 -subunit; 1/0.5 µgfor cortical Na-K-ATPase 1 -subunit; 76/38 µg formedullary NHE3; 0.25/0.125 µg for medullary Na-K-ATPase 1 -subunit; 50/25 µg for medullary - and -ENaC;54/27 µg for cortical -ENaC. NHE3, NaPi2, DPPIV, NCC, NHERF, NKCC,and Na-K-ATPase 1 - and -subunits were detected andquantified with Odyssey Infrared Imaging System (LI-COR). ENaC - and -subunits were detected with enhanced chemiluminescence (Amersham),and autoradiographic signals were quantified with a Bio-Rad imagingdensitometer ( n = 6/group). Table 1 summarizes thedensitometric analysis. * P t -test.& p8 ]/ B4 \# c+ V/ J3 i
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Table 1. Densitometric analysis of immunoblots for sodium transporters
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DISCUSSION* J8 y4 b) h/ t) {
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Ye and Campese ( 8, 33, 34 ) have characterized manyfeatures of this phenol injury model that support a role for central and renal sympathetic nervous system (SNS) activation in the genesis and maintenance of hypertension. In brief, the injection of 50 µl10% phenol into the cortex of one kidney leads to an immediate elevation of norepinephrine secretion from the posterior hypothalamus, a rise in blood pressure, and an increase in plasma norepinephrine level. Renal denervation before phenol injection prevents the increasein both blood pressure and norepinephrine secretion from posteriorhypothalamus. Sympathetic activity recorded directly from renal nervesincreases after phenol injection, and the -adrenergic-receptor blocker phentholamine normalizes blood pressure ( 35 ).Five weeks after phenol injection, the site of injection is reduced toa microscopic scar, yet hypertension and elevated norepinephrine secretion from posterior hypothalamus persist; ablation of the injuredkidney at 4 wk normalizes blood pressure, perhaps due to elimination ofthe renal afferent impulses ( 8 ). These findings allsupport a role for central and renal SNS activation and -adrenergic-receptor activation in the genesis and maintenance ofhypertension induced by phenol injury. These results complement theevidence supporting a role of SNS activation in the pathogenesis ofhypertension induced by renal diseases, including chronic renal failure(CRF) ( 5, 11, 34 ).$ q( h: V& S: D/ h3 Q8 B
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Our laboratory recently investigated the acute effects of phenol injuryon renal sodium transport ( 32 ). Thirty minutes after phenol injection, NHE3 and NaPi2 were redistributed from theintermicrovillar cleft and intracellular membranes to the apicalmicrovilli and apical alkaline phosphatase activity doubled.Additionally, the responses were prevented by prior denervation. Thesefindings provide the first in vivo evidence that SNS stimulationactivates proximal tubule apical sodium entry by recruiting NHE3 andNaPi2 transporters to the apical surface, responses that may contribute to the generation and maintenance of elevated blood pressure by hampering the pressure-natriuresis response. The results of this studydemonstrate that a single injection of phenol into one kidney can causepermanent hypertension associated with a persistent redistribution ofrenal cortical NHE3, NaPi2, and Na-K-ATPase to the plasma membrane andan increase in alkaline phosphatase activity, rather than a return tothe basal pre-phenol injection pattern or a change in the patternobserved in other models of chronic hypertension (SHR, 2K1C), whereNHE3 is retracted out of the microvilli (see below). In addition, thisstudy demonstrates a significant decrease in NHE3 pool size in therenal cortex 5 wk after phenol injury, an escape phenomenon that wouldcounter the effect of redistributing a larger fraction of sodiumtransporters to the apical cell surface.
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Comparing the acute and chronic proximal tubule responses tohypertension of distinct origins illustrates that an alteration inrenal sodium transport may be causal with regard to some varieties ofhypertension and compensatory in other varieties. The proximal tubulesodium transporter responses in the phenol injury model of chronichypertension are quite distinct from that seen in chronic hypertension in the SHR. In young prehypertensive SHRs, NHE3 and Na-K-ATPase activity in renal cortex are higher vs. in age-matched Wistar-Kyoto (WKY) ( 16, 26 ) or SD rats ( 21 ),suggesting that elevated sodium transport may contribute to thedevelopment of hypertension. These differences in activity disappear inadult SHRs with established hypertension vs. WKY or SD rats( 13, 21 ). Biochemical ( 21 ) and confocalimmunofluorescence ( 36 ) studies reveal that NHE3 islocalized to the apical brush border in young prehypertensive SHRs andthen redistributes to the intermicrovillar cleft and subapicalmembranes as hypertension becomes established in adult SHRs, mimickingthe redistribution in SD rats challenged by acute hypertension( 37, 38 ). NHE3 is similarly retracted in the Goldblatt2K1C model ( 36 ). The retraction of NHE3 as hypertensiondevelops (acutely or chronically) is likely a homeostatic compensationto normalize salt and water balance as well as a key mechanism tostimulate transglomerular feedback. The proximal tubule responses inthe phenol-induced hypertension model are the opposite to that seen inthe SHR. Specifically, the NHE3 distribution patterns are nearlyindistinguishable between the acute phase (30 min) of phenol injury andchronic phase of hypertension (5 wk), demonstrating a persistent shiftof NHE3 to apical microvilli from internal membranes: in the acutephase, the NHE3 percentage in WI is 27.2 ± 4.1 (phenol) vs. 13.1 ± 2% (saline), that in WII isunchanged, and in WIII is 10.8 ± 2 (phenol) vs.22.8 ± 4.8% (saline); after 5 wk, the percentage in WI is 25.3 ± 3 (phenol) vs. 12.7 ± 2.7%(saline), that in WII is unchanged, and in WIII is to 9.1 ± 1.4 (phenol) vs. 18.9 ± 3.4% (saline). NaPi2and DPPIV distribution patterns, as well as alkaline phosphataseactivation, are also similar in acute and chronic phases ofphenol-induced hypertension. The fact that the percentages of NHE3,NaPi2, and DPPIV and the activity of alkaline phosphatase in themicrovilli are persistently increased goes along with the observedpersistence of SNS stimulation. In summary, the responses of proximaltubule transporters to the chronic hypertension in the phenol injury model are the opposite to those seen in the chronic hypertension ofthe SHR and Goldblatt 2K1C models. We speculate that thesignals driving the internalization of NHE3 and NaPi2 duringhypertension per se are overridden by opposing signals, most likely theactivated SNS, which drive transporters to the apical membrane,associated with a significant blunting of the pressure-induced diuresisand natriuresis.
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3 L! D$ U& w6 V5 u9 H8 f, TThere have been many reports of the effects of norepinephrine onproximal tubule sodium transport and transporters in isolated proximaltubules. In brief, norepinephrine stimulates sodium reabsorption andouabain-sensitive rubidium uptake and decreases intracellular sodium,evidence for an increase in plasma membrane Na-K-ATPase number oractivity ( 12 ). Studies of the signaling mechanisms inisolated proximal tubule have shown that -agonist oxymetazoline stimulation of Na-K-ATPase transport activity was prevented by either 1 - or 2 -receptor antagonism( 17 ), and studies in cultured proximal tubule cellsdemonstrate a 2 -adrenoreceptor-mediated increase inNa-K-ATPase transport activity secondary to increased apical sodiumentry ( 29 ). In this study, we observed a redistribution ofNa-K-ATPase activity to WI, where the peak basolateralmembrane Na-K-ATPase resides ( 37 ), and did not measurestimulation of total Na-K-ATPase V max activityin a membrane preparation after phenol injury-induced hypertension.These findings are in agreement with the norepinephrine studies in celland tubules, where transport activity in the membrane was assessed. Thefindings are also in agreement with studies in cultured lung cells,where adrenergic agents have been shown to stimulate Na-K-ATPase viainsertion of Na-K-ATPase from intracellular vesicles to the plasmamembranes ( 2 ). We have previously shown that hypertensionper se decreases Na-K-ATPase in the renal cortex ( 22, 23, 37 ), so it is likely that the Na-K-ATPase activity anddistribution after phenol injury may be the product of the combinedmultiple stimuli of SNS stimulation and hypertension.
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3 n4 m6 m8 v1 f: d) ]Hypertension and other renal pathologies can alter the total abundanceof sodium transporters along the nephron in a pathology-dependent fashion. For example, in certain disorders of extracellular volume expansion, kidneys can escape from sodium retention by downregulating one or more of renal sodium transporters, thereby depressing sodium reabsorption to match sodium excretion to sodium intake. The molecular mechanisms responsible for regulation of sodium transporters along thenephron have been investigated in a number of models ( 30 ). For example, when the aldosterone level is inappropriately elevated, e.g., primary aldosteronism, the reabsorptive activity of ENaCs isincreased while the renal abundance of NCC is profoundly and selectively decreased. This response appears to be the chief molecular mechanism by which the kidney overcomes the sodium-retentive effect ofaldosterone. Similarly, long-term pressure-natriuresis has beenreported to be associated with inhibition of distal tubule sodiumtransporters ( 23 ). In contrast, increased Na-K-ATPase, NCC, and ENaC protein pool sizes are increased in the obese Zucker ratand may be responsible for sodium retention and hypertension ( 3 ). In the present study, phenol injury-induced chronichypertension is associated with a 44% fall in total NHE3 abundance inthe renal cortex. When one considers the fact that NHE3 distribution in WI roughly doubles (from 12.7 ± 2.7 to 25.3 ± 3%), this 44% drop in total cortical NHE3 abundance would return thetotal amount of NHE3 in apical membranes ( WI ) close to thatseen before phenol injection. Therefore, this marked reduction may bethe major molecular mechanism responsible for adaptation to chronicstimulation of SNS activity and may contribute to resetting thepressure-natriuresis relationship. It remains to be determined whetherthis decrease in NHE3 is due to decreased synthesis or increaseddegradation. Proximal tubule NHE3 abundance is also reduced 50% in CRFinduced by 5 6 nephrectomy, a response that may contribute toincreased sodium excretion in CRF ( 19 ). Both NHERF andDPPIV have been reported to directly interact with NHE3 ( 14, 28 ). Despite the fact that the cortical NHE3 pool size decreased44% after 5 wk, there was no detectable decrease in total DPPIV or NHERF, indicating that the decrease in NHE3 was specific and that thereis not a defined stoichiometry between NHE3 and these associated proteins (which are known to have other functions in the cells). Additionally, the drop in NHE3 abundance is region specific as therewas no detectable change in medullary NHE3 abundance. In addition,there was no significant change in loop or distal sodium transporterabundance in renal adaptation to the neurogenic phenol injury-inducedhypertension and thus no evidence for escape regulation beyond theproximal tubule, in contrast to other models of hypertension studied( 18 ).
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In summary, the persistent redistribution of NHE3, NaPi2, andNa-K-ATPase to the plasma membranes may contribute to the generation and/or maintenance of chronic hypertension induced by phenol injury. Inparallel, the decrease in total cortical NHE3 abundance in the chronicphase of phenol-induced hypertension is evidence for a coincidentescape mechanism in the same region of the nephron that couldcounteract the effect of redistributing a larger fraction of sodiumtransporters to the apical cell surface.% y7 c+ k1 [) R+ }" x3 m5 p! A

) z' g; W; U; M; D, Y- y/ P& fACKNOWLEDGEMENTS
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* i, a! V9 W/ n) e# Y# A5 gWe are grateful to Dr. Shaohua Ye for guidance and advice regardingchronic phenol model implementation. Michaela MacVeigh providedassistance in confocal microscopy., ]3 \; U/ e3 o
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昕昕

长时间没来看了 ～～  

张佳

一楼的位置好啊..  

awen

先看看怎么样！  

awen

留个脚印```````  

aakkaa

我仅代表干细胞之家论坛前来支持，感谢楼主！  

nauticus

神经干细胞

xuguofeng

声明一下：本人看贴和回贴的规则，好贴必看，精华贴必回。  

beautylive

拿把椅子看表演

haha3245

站个位在说