Effects of renal denervation on tubular sodium handling inrats with CBL-induced - 《American Journal of Physiology》美国生理学杂志干细胞之家



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作者：Thomas E. N.Jonassen, LoneBrønd, MaleneTorp, MartinGræbe, SørenNielsen, OleSkøtt, NielsMarcussen,  StenChristensen作者单位：1 Department of Pharmacology, University ofCopenhagen, DK-2200 Copenhagen N; The Water andSalt Research Center, Institute of Anatomy, and University Institute of Pathology, Aarhus KommuneHospital, University of Aarhus, DK-8000 Aarhus C; and Department of Physiology and Pharmacology,University of Sou
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      【摘要】' B7 Q4 N' t0 O# h8 R
   This study was designed to examine theeffect of bilateral renal denervation (DNX) on thick ascending limb ofHenle's loop (TAL) function in rats with liver cirrhosis induced bycommon bile duct ligation (CBL). The CBL rats had, as previously shown, sodium retention associated with hypertrophy of the inner stripe of theouter medulla (ISOM) and increased natriuretic effect of furosemide invivo, and semiquantitative immunoblotting showed increased expressionof the furosemide-sensitive Na-K-2Cl cotransporter type 2 (NKCC2) inISOM from CBL rats. DNX significantly attenuated the sodium retentionin the CBL rats, which was associated with normalization of thenatriuretic effect of furosemide, as well as a significant reduction inthe expression of NKCC2 in the ISOM. However, the marked hypertrophy ofthe ISOM found in CBL rats was not reversed by DNX. Together, thesedata indicate that increased renal sympathetic nerve activity known tobe present in CBL rats plays a significant role in the formation ofsodium retention by stimulating sodium reabsorption in the TAL viaincreased renal abundance of NKCC2. ! a! H* ^/ I- M7 Q* _' F4 @
      【关键词】 common bile duct ligation furosemidesensitivesodiumpotassiumchloride cotransporter type thick ascending limb furosemide sodium balance inner stripe of outer medulla. H2 M& `% r/ q; |, e1 l
   INTRODUCTION1 }- h/ b/ r9 S
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IT IS WELL DESCRIBED THAT rats with liver cirrhosis induced by common bile ductligation (CBL) develop edema and ascites. Intact renal innervationseems to play a key role in homeostatic regulatory responses to sodiumdepletion and sodium loading, and it has been suggested that~30-40% of the renal sodium retention during edema-formingconditions such as liver cirrhosis ( 12 ), congestive heartfailure ( 12 ), and nephrotic syndrome ( 18 ) isdependent on intact renal sympathetic innervation. Autoradiographic studies have shown intense norepinephrine labeling throughout the renaltubules ( 4-7 ), and renal sympathetic nerve (RSN)stimulation, which does not affect renal blood flow or glomerularfiltration rate (GFR), causes a reversible decrease in urinary sodiumexcretion in rats ( 39 ). Moreover, free-flow micropunctureand tubular microperfusion studies have shown that this stimulatoryeffect of RSN on tubular sodium reabsorption occurs throughout thetubule ( 11 ) and the magnitude of the stimulatory effect ofRSN seems to be proportional to the density of the renal tubularinnervation, being greatest in the thick ascending limb of Henle'sloop (TAL) and least in the collecting duct ( 4 ). Together,these findings indicate that RSNs directly stimulate renal tubularsodium reabsorption.
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We recently investigated renal function in rats with CBL-induced livercirrhosis ( 20, 22, 24 ) and found that the rats hadincreased natriuretic response to furosemide together with markedhypertrophy of the TAL epithelium in the inner stripe of the outermedulla (ISOM). Moreover, the capacity to increase the medullaryinterstitial sodium concentration in response to thirsting was enhancedin the CBL rats ( 23 ). Together, these observations indicate that increased sodium reabsorption in the TAL plays a significant role in the sodium retention, which eventually will resultin the formation of edema and ascites. Interestingly, recent studiesfrom our own as well as other laboratories show that the furosemide-sensitive Na-K-2Cl cotransporter type 2 (NKCC2) exclusively expressed in the TAL and macula densa is significantly increased inother conditions with sodium retention, such as congestive heartfailure and sepsis ( 21, 32, 35 ).

The present study was therefore designed to examine the effect ofbilateral renal denervation on sodium retention as well as renalfunction and structure, including the expression of NKCC2 and other TALtransporters, the luminal electroneutral sodium-proton exchanger (NHE3)( 9 ), and the basolateral Na-K-ATPase in rats withCBL-induced liver cirrhosis. Renal function was examined inchronically instrumented rats during control conditions and duringacute administration of furosemide. To prevent furosemide-induced sodium and water depletion, we used a computerized servo-controlled sodium- and water-replacement system, where losses of sodium and waterwere replaced momentarily ( 19 ).
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METHODS

Experimental Animals
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Female Wistar rats (230-250 g) from Charles River(Sulzfeld, Germany) were used for the experiments. The animals werehoused in a temperature (22-24°C)- and moisture(40-70%)-controlled room with a 12:12-h light-dark cycle (lighton from 6:00 AM to 6:00 PM). Animals were given free access to tapwater and a diet containing ~140 mmol/kg of sodium, ~275 mmol/kgpotassium, and 23% protein. All animal procedures followed theguidelines for the care and handling of laboratory animals establishedby the Danish government.( q" L& \$ U( R
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Animal Preparation3 z- {) V2 |% l, `5 ^, ?( f
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Cirrhosis was induced by CBL as described by Kountouras andco-workers ( 28 ). Briefly, biliary obstruction inducesportal inflammation and bile duct proliferation, which eventually will result in the formation of cirrhosis. Control rats were subjected tosham-CBL.4 ^% I/ ~. Q) d9 E- l5 n) O( x
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Bilateral renal denervation (DNX) was performed through flankincisions. The adventitia of the renal vein and artery were carefullydissected under a microscope. All visible nerves were cut, and thevessels were coated with 10% phenol in 95% ethanol. With thisprocedure, renal norepinephrine content is reduced to 37 ).% Y8 m% _( F* f7 z" Z5 _

Three weeks after CBL/sham-CBL and renal denervation/sham denervation,permanent medical-grade Tygon catheters were implanted in the femoralartery and vein and a permanent suprapubic bladder catheter wasimplanted in the bladder as described previously ( 22, 38 ).After instrumentation, the animals were housed individually.

Experimental Groups

The experimental groups were as follows: sham (sham-CBL ratswith sham-DNX); sham-DNX (sham-CBL rats with bilateral DNX); CBL (CBLrats with sham-DNX); and CBL-DNX (CBL rats with bilateral DNX).
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The experiments were performed in animals in two series: series 1 ( n = 6-8/group), in which sodium balancestudies were performed, plasma samples for measurement of renin andaldosterone were collected, and kidneys were perfusion fixed; and series 2 ( n = 7-8/group), in whichrenal functions studies were performed, and kidneys were used for immunoblotting.

Series 1
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Sodium balance studies. Four weeks after CBL or sham operation, the rats were placed inmetabolic cages for accurate determination of daily food and waterintake. After 2 days of adaptation, sodium balance was measured dailyfor 3 consecutive days, and the average of the three values was used.Sodium intake was calculated from the amount of diet ingested per24 h, and sodium loss was estimated from the amount of sodiumexcreted in the urine within the same 24 h. Sodium balance wasthen calculated as the difference between sodium intake and sodium excretion.

Measurement of plasma renin and aldosterone. Five weeks after CBL or sham operation (i.e., 2 days after thetermination of the sodium balance studies), the rats were placed inrestraining cages, arterial blood samples (total volume 1.0 ml) weredrawn from a permanent arterial catheter, and the plasma was stored at 20°C for later measurement of renin and aldosterone. Plasma reninconcentration was measured by ultramicroradioimmunoassay of generatedANG I with the "antibody-trapping" technique of Lykkegaard andPoulsen ( 30 ). Aliquots of plasma were diluted 20- to80-fold with Tris buffer containing human albumin, and 5-µl portionsof these samples were incubated for 24 h at 37°C with 20 ml of a reaction mixture that contained purified rat renin substrate (~1,200 ng ANG I equivalents/ml). This incubation was followed byradioimmunoassay of generated ANG I. Plasma renin concentration wasmeasured in reference to renin standards obtained from the NationalInstitute for Biological Standards and Control (Potters Bar, Herts, UK; 1 milli-Goldblatt unit = 160 pg ANGI · ml 1 · h 1 ).Plasma aldosterone concentration was measured by radioimmunoassay usinga commercial kit (Coat-A-Count Aldosterone, DPC, Los Angeles, CA).
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Histological examinations. Then, the rats were anesthetized with halothane-nitrous oxide, and theleft kidney was perfused in vivo for 3 min with 1.5% glutharaldehydein Tyrode's solution with added 2.25% dextran T-40 (perfusionpressure: 150 mmHg) and postfixed in perfusion fluid for laterstereological examination. The kidneys were sliced at a 90° angle onthe longitudinal axis of the kidney. The 2-mm-thick slices wereembedded in paraffin, and 3- to 4-µm-thick sections were cut andstained with hematoxylin-eosin. From this, the volume fractions of thedifferent renal zones were measured stereologically ( 17 ).All sections were investigated by light microscopy point counting(using a stage motor), in systematic order with random starting points.The number of points hitting within each zone was estimated. The totalnumber of hitting points within each kidney slice was 200-300, andeach field of vision included a grid with 4 points. Data from all2-mm-thick slices were included, which means that approximately five toseven sections from each kidney were examined. When the volumefractions of the different zones are known, the absolute volumes of thezones can be calculated by multiplying the volume fractions with thevolume of the kidney (equal to the kidney weight, assuming that thespecific gravity of the kidney is 1 g/cm 3 )( 22 ).# Q1 W2 `# `! l2 D
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Renal clearance studies. Renal hemodynamic and tubular responses to furosemide were examined byclearance techniques in conscious, chronically instrumented rats 4 wkafter a CBL/sham-CBL operation. Before the clearance experiments, allrats were adapted to the restraining cage used for these experiments bytraining them for two periods of 2 h each on consecutive days.Clearance experiments were started at 8:00 AM. Clearance of[ 14 C]tetraethylammonium bromide was used as a marker foreffective renal plasma flow, clearance of [ 3 H]inulin as amarker for GFR, and clearance of lithium (C Li ) as a markerfor distal delivery ( 42 ). In addition to minor amounts oflithium in the infusion solutions, lithium was added to the diet (12 mmol/kg diet) for 3 days before the experiments to avoid acute effectsof lithium on renal function ( 29 ). The clearance experiments were performed as follows. Clearance markers in 150 mMglucose, 13 mM NaCl, and 3 mM LiCl were infused at a constant rate of2.5 ml/h throughout the experiments. After a 90-minequilibration period where steady-state levels of the tracer substanceswere reached, urine was collected in 2 × 30-min control periodsto characterize baseline values of systemic and renal hemodynamics andtubular function. Then, infusion of furosemide was started at aconstant rate of 0.50 mg/h, and urine was collected in 8 × 30-minperiods during furosemide infusion. To avoid sodium and waterdepletion, all furosemide-induced water and sodium losses wereimmediately replaced using a computerized servo-controlled water- andsodium-replacement system ( 19, 28 ), which originally wasdeveloped by Andersen and Bie ( 2 ) for sodium and water replacement in dogs. The servo system consists of a sodium-sensitive electrode (Radiometer) that continuously measures sodium concentration in the urine, a balance (Sartorius model MC 1, Göttingen,Germany) that registers urine production (integration period: 1 min),and two infusion pumps (model 200-7103, Harvard Apparatus) thatinfuse 50 mM glucose and 300 mM NaCl, respectively. The input from the electrode and the balance is analyzed on-line in Lab-View, and theinfusion rate of the two infusion pumps is continuously altered toreplace the urinary water and sodium losses. The accuracy of thissystem lies within the micromolar range for urinary sodium concentration between 10 and 150 mM. Arterial blood samples (300 µleach) were collected into ammonium-heparinized capillary tubes at theend of the equilibration period, at the end of the control period, andonce every hour during furosemide infusion. All blood samples wereimmediately replaced with heparinized blood from a donor rat. Meanarterial pressure (MAP) was measured throughout. Electrolytes insamples of plasma and urine were determined by atomic absorptionspectrometry, using a PerkinElmer series 2380 and a PerkinElmer Analyst300 atomic absorption spectrometer. [ 14 C]tetraethylammonium and [ 3 H]inulin weredetermined by double-label scintillation counting in a Packard 2250 CAliquid scintillation counter. Renal clearances (C) and fractionalexcretions (FE) were calculated by the standard formula1 A$ n9 z$ i4 e) I) [
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where U is concentration in urine, V is urine flow rate, and Pis plasma concentration.
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Semiquantitative immunoblotting. Five days after the renal clearance studies, the rats were anesthetizedwith halothane/N 2 O, and the right kidney was removed, immediately frozen in liquid nitrogen, and stored at 80°C until processing for membrane fractionation. The outer medulla was isolated and homogenized using a tissue homogenizer (Ultra-Turrax T8, Ika, Staufen, Germany) in 10 ml of ice-cold homogenizing buffer containing 300 mM sucrose, 25 mM imidazol, 1 mM EDTA-disodium salt, and the following protease inhibitors: Pefabloc (0.1 mg/ml buffer) and leupeptin (4 µg/ml buffer); and phosphatase inhibitors sodium orthovanadate (184 µg/ml), sodium fluoride (1.05 mg/ml), and okadeic acid 82 (ng/ml), pH adjusted to 7.2 with 0.1 M HCl. Proteinconcentration in the homogenate was measured by use of a commercial kit(Pierce BCA Protein Assay Reagent Kit, Pierce, Rockford, IL). Allsamples were then diluted to a final protein concentration of 4 µg/µl with the additon of sample buffer (in the final solution: 486 mM Tris · HCl, pH 6.8, 7% glycerol, 104 mM SDS,0.0875 mM bromphenol blue), dithiothreitol (25 mM in thefinal solution), and homogenizing buffer. The samples were thensolubilized at 60°C for 10 min.

Samples of homogenates were run on 7.5% polyacrylamide minigels. Theproteins were then transferred by electrophoresis from the gels to PVDFmembranes (90 min, 100 V). After 60-min blocking in 5% milk in PBS-Tbuffer, membranes were probed overnight at 4°C with the appropriateprimary antibody. For measurement of NKCC2, we used a rabbit polyclonalanti-NKCC2 antibody raised to a synthetic peptide corresponding toamino acids 33-55 of rat NKCC2 ( 14 ). For measurementof NHE3, we used a rabbit polyclonal anti-NHE3 antibody raised againsta synthetic peptide corresponding to amino acids 809-831 of ratNHE3 ( 36 ). For measurement of Na-K-ATPase, we used acommercial rabbit polyclonal anti- 1 -subunit antibody(06-520; Upstate Biotechnology, Lake Placid, NY). Finally, formeasurement of aquaporin-2 (AQP2), we used a commercialaffinity-purified goat polyclonal antibody (sc-9882; Santa CruzBiotechnology, Santa Cruz, CA).& {1 @. H( T, m, u! H' d0 m

The labeling was visualized with horseradish peroxidase-conjugatedsecondary antibody [P448 (rabbit) or P0449 (goat); Dako; diluted1:3,000] using an enhanced chemiluminescence system (ECL , Amersham).The broad ~165-kDa band corresponding to NKCC2, the ~86-kDa bandcorresponding to NHE3, the ~96-kDa band corresponding to Na-K-ATPaseor the 29- and 35- to 50-kDa bands corresponding to AQP2 andglucosylated AQP2, respectively, were scanned by use of a FluorXmulti-imager (Bio-Rad Laboratories). Densitometry of individual bandswas quantitated using the software program Quantity One, version 4.2.3 (Bio-Rad Laboratories). Protein labeling in samples from the differentgroups was expressed relative to the mean expression of the controlmaterial run on the same gel.1 a) l) V* \2 C$ q5 k: [8 w
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Statistics

Data are presented as means ± SE. To evaluate the effectof furosemide, the average values during the two 30-min control periods were compared with the average values during the last three 30-min periods of the furosemide-induced diuresis. Comparisons were performed by two-way analysis of variance followed by Fisher's least significant difference test. Differences were considered significant at the 0.05 level.' E+ n* z8 j* _, Y

RESULTS8 @9 J/ j2 u: T. |5 Y

Daily sodium intake was similar in all groups, but daily sodiumexcretion significantly decreased in the CBL rats, which indicated thepresence of sodium retention relative to the sham-operated controlanimals (Table 1 ). DNX had no effect onsodium handling in the sham-operated control rats, but sodium retentionwas significantly attenuated in CBL-DNX rats (Table 2 ). Twenty-four-hour urine production andurine osmolality were similar in the sham and CBL rats. DNX had noeffect on urine production or urine osomolality in the sham-operatedrats, but in CBL rats DNX significantly increased the production ofsolute-free urine. Plasma levels of renin, aldosterone, sodium, andpotassium as well as plasma osmolality were similar in all four groups(Table 2 ).# E. j1 `) T1 u! h; {) x, C

Table 1. Twenty-four-hour urine production, sodium intake, sodium excretion, andsodium balance in rats 5 wk after liver cirrhosis was induced by commonbile duct ligation9 {$ L6 c/ L6 u% r1 \1 d
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Table 2. Body weight and plasma levels of renin and aldosterone in rats 5 wkafter liver cirrhosis was induced by common bile duct ligation: D0 x. \& L+ X! i, R+ L  b0 t4 M
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Renal Function Studies

Baseline values. MAP and effective renal plasma flow (ERPF) were similar in all fourgroups (Table 3 ). GFR was decreasedcompared with controls, and the effective filtration fraction wastherefore, as previously shown ( 22, 23, 25 ), significantlydecreased in the CBL rats. DNX had no significant effects on effectivefiltration fraction or GFR in either CBL or sham-CBL rats. There wereno significant differences in C Li between the CBL andsham-CBL groups, whereas DNX increased C Li in both the CBLand the sham-CBL rats. Baseline values for V and urinary sodiumexcretion rate (U Na V) were similar in allgroups 1 (data not shown).0 }5 k( Q0 M  O  p
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Table 3. Effects of bilateral renal denervation on mean arterial pressure,effective renal plasma flow, glomerular filtration rate, effectivefiltration fraction, lithium clearance, and fractionallithium excretion in rats 5 wk after liver cirrhosis was induced bycommon bile duct ligation
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Effect of furosemide on renal sodium handling. Constant furosemide infusion under conditions in which water and sodiumdepletion was prevented by use of a computerized servo-controlled system induced a prolonged and sustained diuretic and natriuretic response in all four groups. Within all the groups, diuresis and natriuresis reached a steady state after an ~150-min infusion (Fig. 1 ). The furosemide-induced natriuresiswas as previously shown ( 20, 22-24 ) to besignificantly increased in the CBL rats [ U Na V (CBL:22.9 ± 3.1 vs. sham: 13.4 ± 2.0 µmol · min 1 · 100 g body wt 1, P FE Na (CBL: 20.2 ± 2.6 vs. sham: 10.2 ± 1.7%, P 0.01)]. Similarly, the change infractional distal sodium excretion (C Na /C Li )was significantly increased in the CBL rats[ C Na /C Li (CBL: 30.8 ± 3.9 vs. sham:19.8 ± 2.4%, P thenatriuretic response to furosemide in CBL rats [ U Na V(CBL-DNX: 14.6 ± 2.1 vs. CBL: 22.9 ± 3.1 µmol · min 1 · 100 g body wt 1, P FE Na (CBL-DNX: 11.7 ± 2.6 vs. CBL: 20.2 ± 2.6%, P C Na /C Li (CBL-DNX: 19.3 ± 2.9 vs CBL: 30.8 ± 3.9%, P natriuretic response in the sham-CBL rats.Furosemide produced similar changes in MAP, GFR, ERPF, andFE Li in all four groups (data not shown).$ k+ f  U; J, C4 \
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Fig. 1. Effect of continuous furosemide infusion in conscious chronicallyinstrumented rats, wherein all sodium and water losses were momentarilyreplaced by use of a computerized servo-controlled sodium andwater-replacement system. The rats were subjected to common bile ductligation (CBL) or sham operation (sham) 5 wk earlier and eitherunderwent bilateral renal denervation (DNX) or were sham denervated atthe time of CBL/sham-CBL. A : furosemide-induced increase inurine flow rate ( V). B : furosemide-induced increase inurinary sodium excretion rate ( U Na V). C :furosemide-induced increase in fractional sodium excretion( FE Na ). D : furosemide-induced increase infractional distal sodium excretion( C Na /C Li ). Values are means ± SE; n = 7-8 rats/group. BW, body wt.* P # P6 G' X; v7 F- R& G# L* ~' W4 a
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Renal histopathology. The stereological analysis performed on the in vivo perfusion-fixedkidneys showed, in accordance with previous reports from our laboratory( 20, 22 ), that CBL rats had marked and selective hyperthrophy of the ISOM (absolute volume: 154 ± 17 vs. 227 ± 14 mm 3, P in CBL rats(Fig. 2 ).0 X$ B) ]* y5 O, u  [. j1 ?2 O

Fig. 2. Absolute volumes of the renal zones from rats subjected to CBL orsham-operation (sham) 5 wk earlier and that underwent either DNX orsham at the time of CBL/sham-CBL. A - D :cortex, inner stripe of outer medulla, outer stripe of outer medulla,and inner medulla, respectively. Values are means ± SE; n = 7-8 rats/group. * P

Expression of sodium transporters in renal outer medulla. Figure 3 A shows an immunoblotof membrane fractions (40 µg protein/lane) from renal outer medullarypreparations. The affinity-purified anti-NKCC2 antibody recognizes abroad band at ~165 kDa corresponding to the furosemide-sensitivetype-2 Na-K-2Cl cotransporter exclusively expressed in the TAL and inthe macula densa ( 14 ). Densitometry of all samples (Fig. 3 B ) revealed an increased expression of NKCC2 in the outermedulla in CBL rats compared with sham-operated controls (sham:100 ± 14 vs. CBL: 131 ± 4%, P decreased the expression of NKCC2 in CBL rats, asshown in Fig. 3, C and E. Results of thedensitometry were CBL: 100 ± 15 vs. CBL-DNX: 57 ± 11%, P ± 21%, not significant. DNX had no effect on NKCC2 expression in the sham-DNX rats (data not shown). Figure 4 A shows an immunoblot ofmembrane fractions (40 µg protein/lane) from renal outermedullary preparations. The affinity-purified anti-NHE3antibody recognizes a band at ~86 kDa corresponding to NHE3 expressedin the TAL and in the proximal tubules ( 9 ). Densitometryof all samples (Fig. 4 B ) revealed a decreased expression ofNHE3 in the outer medulla of CBL rats compared with sham-operatedcontrols (sham: 100 ± 8 vs. CBL: 57 ± 4%, P in Fig. 4, C - F, no effect on the expression of NHE3 inCBL rats. Similarly, DNX had no effect on NHE3 expression in thesham-DNX rats (data not shown).
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Fig. 3. Immunoblots of membrane fractions (40 µg protein/lane)from a renal outer medullary preparation. The rats were subjected toCBL or sham operation (sham) 5 wk earlier and either underwent DNX orwere sham denervated at the time of CBL/sham-CBL. The immunoblots werereacted with affinity-purifiedanti-Na -K -2Cl cotransporter(NKCC2) and reveal a band at ~165 kDa. A and B :immunoblot showing sham vs. CBL and densitometry performed on allsamples, respectively. C and D : immunoblotshowing CBL vs. CBL-DNX and densitometry performed on all samples,respectively. E and F : immunoblot showing shamvs. CBL-DNX and densitometry performed on all samples, respectively.Values are means ± SE. * P # P
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Fig. 4. Immunoblots of membrane fractions (40 µg protein/lane)from a renal outer medullary preparation. The rats were subjected toCBL or sham operation (sham) 5 wk earlier and either underwent DNX orwere sham denervated at the time of CBL/sham-CBL. The immunoblots werereacted with affinity-purified anti-Na -H cotransporter (NHE3) and reveal a band at ~86 kDa. A and B : immunoblot showing sham vs. CBL and densitometryperformed on all samples, respectively. C and D :immunoblot showing CBL vs. CBL-DNX and densitometry performed on allsamples, respectively. E and F : immunoblotshowing sham vs. CBL-DNX and densitometry performed on all samples,respectively. Values are means ± SE. * P
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We also measured the abundance of the 1 -subunit of theNa-K-ATPase in the outer medulla. Na-K- ATPase is expressed in both the TAL and the collecting ducts. All our measurements showed that theabundance of the 1 -subunit was similar in sham and CBL rats and that DNX had no effect on the expression level in either shamor CBL rats (data not shown).
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Expression of AQP2 in the renal outer medulla. Finally, we measured the expression level of AQP2 protein in the outermedulla. AQP2 is expressed in the principal cells of the collectingducts, and the affinity-purified anti-AQP2 antibody recognizes the29-kDa and the 35- to 50-kDa band, corresponding to nonglycosylated andglycosylated AQP2 protein, respectively. In accordance with previousreports from our laboratory ( 23, 25 ), AQP2 expression wassignificantly decreased in CBL rats (Fig. 5, A and B ). As shown in Fig. 5, C - F, renal denervation had noeffect on the expression of AQP2 in CBL rats. Similarly, the expressionof AQP2 was unchanged in the sham-DNX rats (data not shown).% I1 k! }# T& _; b2 @" I% K
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Fig. 5. Immunoblots of membrane fractions (20 µg protein/lane)from a renal outer medullary preparation. The rats were subjected toCBL or sham operation (sham) 5 wk earlier and either underwent DNX orwere sham denervated at the time of CBL/sham-CBL. The immunoblotswere reacted with affinity-purified anti-aquaporin-2 (AQP2) and reveal29-kDa and 35- to 50 kDa bands, corresponding to nonglycosylated andglycosylated AQP2 protein, respectively. A and B :immunoblot showing sham vs. CBL and densitometry performed on allsamples, respectively. C and D : immunoblotshowing CBL vs CBL-DNX densitometry performed on all samples,respectively. E and F : immunoblot showing shamvs. CBL-DNX and densitometry performed on all samples, respectively.Values are means ± SE. * P
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The major finding of the present study is that DNX ameliorates thedevelopment of renal tubular dysfunction in rats with CBL-induced livercirrhosis by mechanisms most probably involving inhibition of increasedsodium reabsorption in the TAL. The sodium balance studies revealedthat DNX normalized 24-h sodium balance, and the clearance studiesshowed that DNX normalized the increased natriuretic response tofurosemide found in CBL rats. Moreover, DNX significantly reduced theexpression of NKCC2. However, the marked hypertrophy of the ISOM foundin CBL rats was not reversed by DNX.

An increasing number of studies have evaluated TAL function inconditions with impaired renal sodium handling. The present studyconfirmed previous findings from our own laboratory ( 20, 22-24, 26 ) indicating that sodium reabsorption in the TALis increased in cirrhotic rats and plays a significant role in the sodium retention that eventually results in the formation of edema andascites. However, not only liver cirrhosis seems to be associated withaltered TAL function. Increased NKCC2 expression has also been found inrats with congestive heart failure ( 32, 35, 41 ), andrecently we have shown that sepsis-induced acute renal failure isassociated with increased NKCC2 expression ( 26 ). Moreover,Alvarez-Guerra and Garay ( 1 ) have reported increased natriuretic effect of bumetanide associated with increasedbumetanide-sensitive rubidium uptake in TAL from Dahl-S hypertensiverats, and Manning and co-workers ( 31 ) have recently shownincreased NKCC2 expression in rats with prenatally programmedhypertension induced by a maternal low-protein diet during pregnancy.Together, these data seem to support a role of regulation of NKCC2abundance in a number of pathophysiological conditions with impairedrenal sodium handling.
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RSNs are important modulators of renal sodium excretion through releaseof the neurotransmitter norepinephrine. DiBona and co-workers( 13 ) have demonstrated that RSN activity is increased during conditions with extracellular volume expansion, including livercirrhosis and congestive heart failure ( 13 ). Moreover, long-term sodium balance studies have shown that renal denervation significantly attenuates the development of excess sodium accumulation in rats with liver cirrhosis or congestive heart failure( 12 ). Several segments of the nephron are closelyassociated with sympathetic neuronal varicosities( 4-7 ), and the highest number of neural fibers pertubule is found in the TAL ( 7 ). The TAL possesses both 2 ( 33 )- and -adrenergic receptors( 16 ), and the selective -adrenergic receptor agonistisoproterenol increases sodium reabsorption in the TAL( 3 ). Thus renal nerve activity seems to be involved in theregulation of sodium reabsorption in TAL. In the present study,bilateral DNX prevented the excess sodium retention in CBL rats. Thisnormalization of the sodium balance was associated with a significantlyreduced expression of NKCC2 in the outer medulla in CBL rats and withnormalization of the natriuretic response to furosemide. Together,these findings indicate that increased RSN activity known to be presentin CBL rats plays a significant role in the formation of sodiumretention by stimulating sodium reabsorption in the TAL.
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We also examined the expression of NHE3 in the outer medulla. NHE3,which plays a major role in the regulation of urine acidification andmight work as an alternative route for TAL sodium reabsorption, wassignificantly decreased in CBL rats. The mechanism behind this findingis unknown. However, DNX had no effect on the expression of NHE3 inboth sham and CBL rats, indicating that changes in the abundance ofthis transporter are not affected by RSN.

CBL rats had, as was previously shown ( 20, 22 ), markedhypertrophy of the ISOM. Similar morphological changes are found inrats chronically treated with vasopressin ( 8, 10 ), and wehave shown that this hypertrophy is absent in vasopressin-deficient Brattleboro rats with CBL-induced liver cirrhosis ( 22 ).Moreover, we have shown that chronic treatment with the somatostatinanalog octreotide prevents the development of ISOM hypertrophy in CBL rats by an unknown mechanism ( 22 ). Despite the markedeffect of DNX on TAL function in CBL rats, DNX did not prevent theformation of ISOM hypertrophy, which strongly indicates that RSNstimulation has functional but not hypertrophic action in the TAL.- W1 b; i0 T; K3 F! I

Renal denervation increased the formation of solute-free urine in CBLrats. The final regulation of urine production is regulated byvasopressin and depends on 1 ) expression and membranetargeting of AQP2 in the collecting ducts and 2 ) themagnitude of the corticomedullary osmotic gradient generated by sodiumreabsorption in the TAL. As was previously shown ( 23, 25 ),CBL rats had significantly decreased expression of AQP2. DNX had noeffect on the abundance of AQP2 in either normal or CBL rats. However,because DNX prevented the formation of increased TAL sodiumreabsorption, the formation of an increased corticomedullary osmoticgradient ( 21 ) most probably was prevented as well,resulting in the production of an increased amount of solute-free urine.6 w6 U1 P1 Y8 c6 a
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In summary, the present data indicate that RSN activity plays asignificant role in the formation of sodium retention in CBL rats bystimulating sodium reabsorption in the TAL. An increasing number ofreports support the hypothesis that regulation of TAL sodiumreabsorption, including NKCC2 abundance, plays a significant role indifferent pathophysiological conditions with impaired renal sodiumhandling ( 1, 21, 31, 32, 35, 41 ). Detailed studies of therole of RSNs for the regulation of TAL function in thesepathophysiological conditions, which include congestive heart failure,hypertension, and sepsis, are warranted.
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ACKNOWLEDGEMENTS

The technical assistance of Iben Nielsen, Barbara Seider, and HayaHolmegaard is acknowledged.
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