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作者：ChunlingLi, WeidongWang, Tae-HwanKwon, Mark A.Knepper, SørenNielsen, JørgenFrøkiær,作者单位：1 The Water and Salt Research Center and Department of Cell Biology, Institute of Anatomy,University of Aarhus, DK-8000 Aarhus C; Institute ofExperimental Clinical Research and Department ofClinical Physiology, Aarhus University Hospital-Skejby, University ofAarhus, DK-8200 Aarhus N, Denmark; Depart " ]( r3 D- V m, w% p
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; y8 P+ j9 T% I( h( K7 G* @ 【摘要】
/ A5 F- E) F9 a) D1 K9 K" {" ]# H It has been demonstrated previouslythat ureteral obstruction was associated with downregulation of renalAQP2 expression and an impaired urinary concentrating capacity (Li C,Wang W, Kwon TH, Isikay L, Wen JG, Marples D, Djurhuus JC, Stockwell A, Knepper MA, Nielsen S, and Frøkiær J. Am J Physiol RenalPhysiol 281: F163-F171, 2001). In the present study, changesin the expression of major renal Na transporters were examined in a ratmodel with 24 h of unilateral ureteral obstruction (UUO) toclarify the molecular mechanisms of the marked natriuresis seen afterrelease of UUO. Urine collection for 2 h after release of UUOrevealed a significant reduction in urinary osmolality, solute-freewater reabsorption, and a marked natriuresis (0.29 ± 0.03 vs.0.17 ± 0.03 µmol/min, P reductions in the abundanceof major renal Na transporters: type 3 Na /H exchanger (NHE3; 24 ± 4% of sham-operated control levels), type 2 Na-P i cotransporter (NaPi-2; 21 ± 4%), Na-K-ATPase(37 ± 4%), type 1 bumetanide-sensitive Na-K-2Cl cotransporter(BSC-1; 15 ± 3%), and thiazide-sensitive Na-Cl cotransporter(TSC; 15 ± 4%). Immunocytochemistry confirmed the downregulationof NHE3, BSC-1, and TSC in response to obstruction. In nonobstructedcontralateral kidneys, a significant reduction in the abundance ofinner medullary Na-K-ATPase and cortical NaPi-2 was found. This maycontribute to the compensatory increase in urinary production (23 ± 2 vs. 13 ± 1 µl · min 1 · kg 1 )and increased fractional excretion of urinary Na (0.62 ± 0.03 vs.0.44 ± 0.03%, P major renal Na transporters in rats with UUO maycontribute to the impairment in urinary concentrating capacity andnatriuresis after release of obstruction, and reduced levels ofNa-K-ATPase and NaPi-2 in the contralateral nonobstructed kidney maycontribute to the compensatory increase in water and Na excretion fromthat kidney during UUO and after release of obstruction.
# a. q4 B2 \" H& T 【关键词】 thick ascending limb of Henle‘s loop collecting duct proximaltubule distal convoluted tubule obstructive nephropathy sodiumexcretion
s; h$ H! L7 {9 Q INTRODUCTION
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* y. `/ \9 f# d: F! T" b" {URINARY TRACT OBSTRUCTION impairskidney function and is characterized by profound changes in renalhemodynamics and tubular functions. In particular, the renal tubularfunctions that regulate body fluid and electrolyte homeostasis arecompromised. It is well established that urinary tract obstruction isassociated with a marked reduction in the ability to concentrate urine.We have demonstrated previously that ureteral obstruction is associated with downregulation of renal aquaporins (AQPs)(15, 16, 30). In addition, urinary tract obstruction may alter renaltubular transport of electrolytes due to dysregulation of key renalsodium transporters ( 21, 32, 36 ).
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3 ^4 }# r7 }" p" X. MUrinary concentration and dilution depend on a discrete segmentaldistribution of transport properties along the renal tubule. Urinaryconcentration depends on 1 ) active solute reabsorption inthe thick ascending limb (TAL) of Henle's loop to generate a highosmolality in the medullary interstitium, as a consequence ofcountercurrent multiplication, and 2 ) a high waterpermeability (constitutive or vasopressin regulated) of renal tubules,which chiefly depends on AQPs. Previously, in vitro studies of thetransporter of ions in specific segments of the nephron and in vivomicropuncture studies have demonstrated an impairment of fluid andsodium reabsorption in the proximal tubule, the medullary TAL (mTAL),the distal convoluted tubule (DCT), and the collecting duct (CD) afterobstruction ( 21 ), consistent with a marked urinaryconcentrating defect and salt wasting after ureteral obstruction( 15, 16, 30 ). However, a thorough understanding of whichsodium transporters along the nephron are involved at the molecularlevel has yet to be achieved.& u6 \: _. G2 M0 F
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Recently, we demonstrated that altered expression of major renalsodium transporters is associated with deranged urinary concentration and urinary sodium excretion in several conditions with water andsodium balance disorders ( 4, 29, 46, 47 ). The proximal tubule is the main site for renal tubular sodium reabsorption, and thetype 3 Na /H exchanger (NHE3) is mainlyresponsible for apical sodium reabsorption ( 2 ), and thetype 2 Na-P i cotransporter (NaPi-2) is also involved ( 3, 31 ). Downregulation of NHE3 and NaPi-2 has beendemonstrated in several conditions known to have proximal tubuledefects in sodium reabsorption and increased urinary sodium excretion( 2, 29, 46 ). In addition, unilateral ureteral obstruction(UUO) is associated with significant changes in renal phosphateexcretion ( 37 ). Moreover, the Na-K-ATPase is heavilyexpressed in the basolateral plasma membrane of the renal tubule cellsand is responsible for sodium reabsorption ( 24 ). Inanimals with acute UUO for 18-24 h, Na-K-ATPase activity waspreviously shown to be markedly decreased in all nephron segments( 21, 22, 39 ). Thus reduced expression levels of NHE3,NaPi-2, and Na-K-ATPase in the obstructed kidney could be speculated tobe associated with the marked natriuresis in response to obstructive nephropathy.
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. f, W9 {0 i3 ^! A1 ]4 I0 UThe key sodium transporters responsible for the active transportof NaCl in the mTAL are the apical bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1 or NKCC2) ( 11, 23, 26, 33, 49 ), NHE3,and basolateral Na-K-ATPase. Particularly, the apically expressedNa-K-2Cl cotransporter in the TAL is known to be regulated byvasopressin and involved in the long-term regulation of the countercurrent multiplication system. Previously, measurement of thesaturable [ 3 H]bumetanide binding to the Na-K-2Clcotransporter indicated that the abundance of Na-K-2Cl is reduced inresponse to UUO ( 21 ). In the DCT, the thiazide-sensitiveNa-Cl cotransporter (TSC or NCC) is chiefly involved in apical sodiumreabsorption ( 27, 34 ). Thus dysregulation of TSC is alsohypothesized to participate in the deranged renal sodium and waterexcretion in response to UUO.
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The purposes of the present study are therefore to characterize themechanisms responsible for salt wasting in obstructive nephropathy 1 ) to examine the changes in renal water and electrolyte handling in rats with a 24-h period of UUO and 2 ) to examinewhether these changes are associated with altered expression of major renal sodium transporters in both the obstructed and the nonobstructed kidneys of rats with UUO.
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/ }4 x' y: Y% [ Q. _+ ZMETHODS
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& V5 z( `8 a0 w% m) p, h, e* OExperimental Animals; v7 f9 e. M" F
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Studies were performed in male Munich-Wistar rats initiallyweighing 250 g (Møllegard Breeding Centre, Eiby, Denmark). The rats were maintained on a standard rodent diet (Altromin, Lage, Germany) with free access to water. During the entire experiment, ratswere kept in individual metabolic cages, with a 12:12-h artificial light-dark cycle, a temperature of 21 ± 2°C, and humidity of55 ± 2%. The rats were allowed to acclimatize to the cages for5-7 days before surgery. The rats were anesthetized with halothane (Halocarbon Laboratories), and during surgery they were placed on aheated table to maintain rectal temperature at 37-38°C. Through a midline abdominal incision, the left ureter was exposed and themidportion of ureter was occluded by a 5-0 silk ligature. Twenty-fourhours later, the rats were either killed ( protocol 1 ) orreleased by inserting polyethylene tubing (PE-35) into the proximalureters to collect urine from the left and right ureter ( protocol2 ), respectively. After urine collection for at least 2 h,the rats were killed. Age- and time-matched sham-operated controls wereprepared and observed in parallel with each UUO group (Fig. 1 ).
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1 w, {' T6 V0 a& P% ^Fig. 1. Diagram of the study design. Protocol 1 :the left ureter was occluded for 24 h in Wistar rats withunilateral ureteral obstruction (UUO), and age-matched sham-operatedcontrol rats were prepared in parallel. Protocol 2 : ratswere prepared similar to protocol 1 with UUO andsham-operated controls. After 24 h, the obstruction was releasedand urine was collected from each ureter for 2 h. n,No. of rats.
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Protocols4 l5 E6 g/ N4 A: M
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The rats were allocated to the following protocols.
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7 w \$ P5 r4 g3 wProtocol 1. Rats underwent UUO for 24 h ( n = 11). Both kidneyswere removed and separately prepared for semiquantitativeimmunoblotting ( n = 7) or immunocytochemistry( n = 4). A sham-operated group was also used( n = 10). R, f% M4 j4 [4 n' O2 e0 |' e: `
( q9 ^$ Q7 [+ i% v# x$ gProtocol 2. Rats underwent UUO for 24 h followed by release, and animals wereobserved during the next 2 h ( n = 8). Urine wascollected for 2 h. A sham-operated group was also used( n = 8).) y- R" o% y6 }$ D$ ^: E
+ N. `" L( n7 rClearance Studies/ Y9 ^$ b8 i1 x- v% D& V' q
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Urine was collected during 24-h periods throughout the study orfor 2 h after the release of obstruction. Clearance studies wereperformed over the last 24 h in protocol 1 or for2 h after release in protocol 2. At the end of eachprotocol, under anesthesia, 2-3 ml of blood were collected into aheparinized tube for determination of plasma electrolytes andosmolality before the rats were killed. The plasma concentrations ofsodium, potassium, creatinine, urea, and phosphate and the urinaryconcentration of creatinine and urea were determined (Vitros 950, Johnson & Johnson). The concentrations of urinary sodium and potassiumwere determined by standard flame photometry (Eppendorf FCM6341).Urinary phosphate was determined (Vitros 250, Johnson & Johnson). Theosmolality of urine and plasma was determined by freezing-pointdepression (Advanced Osmometer, model 3900, Advanced Instruments,Norwood, MA, and Osmomat 030-D, Gonotec, Berlin, Germany).7 G. Z5 E/ x" r
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Membrane Fractionation for Immunoblotting5 M: K: B5 h z" \5 U
! i" n- P8 Z4 e& l3 e' [For removal of kidneys, rats were anesthetized with halothane.The kidney was split into cortex plus outer medulla and inner medullaand frozen in liquid nitrogen. Tissue (inner medulla or cortex plusouter medulla) was finely minced and then homogenized in 1 ml (innermedulla) or 8 ml (cortex plus outer medulla) of dissecting buffer (0.3 M sucrose, 25 mM imidazole, and 1 mM EDTA, pH 7.2, containing theprotease inhibitors 8.5 µM leupeptin and 1 mM phenylmethylsulfonylfluoride) with 5 strokes of a motor-driven IKA homogenizer at 1,250 rpm. The homogenate was centrifuged in a Universal 30 RF centrifuge at4,000 g for 15 min at 4°C. Gel samples (in Laemmli samplebuffer containing 2% SDS) were made from this membrane preparation.
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9 j6 q3 X- k3 \9 V2 C; L8 DElectrophoresis and Immunoblotting; d# m# L% @8 a. K9 B, v' E
- c$ W& k" o) i1 W7 lSamples of membrane fractions from inner medulla or cortexplus outer medulla were run on 12 or 6-16% gradientpolyacrylamide minigels (Bio-Rad Mini Protean II). For each gel, anidentical gel was run in parallel and subjected to Coomassie staining( 43 ). The Coomassie-stained gel was used to ascertainidentical loading or to allow for potential correction for minordifferences in loading after scanning and densitometry of major bands(see below). The other gel was subjected to blotting. After transfer byelectroelution to nitrocellulose membranes, blots were blocked with 5%milk in 80 mM Na 2 HPO 4, 20 mMNaH 2 PO 4, 100 mM NaCl, 0.1% Tween 20, pH 7.5, for 1 h and incubated with primary antibodies (see below)( 10 ) overnight at 4°C. After being washed as above, theblots were incubated with horseradish peroxidase-conjugated secondaryantibody (P447 or P448, diluted 1:3,000, DAKO, Glostrup, Denmark), and bands were visualized using the enhanced chemiluminescence system (ECL;Amersham International). Controls were made with an exchange of theprimary antibody for an antibody preabsorbed with immunizing peptide(100 ng/40 ng IgG) or with preimmune serum (diluted 1:1,000). Allcontrols were not labeled.
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Primary Antibodies( r1 j/ [! X1 h' e
+ o5 o m6 T7 @; ^) N8 mFor semiquantitative immunoblotting and immunocytochemistry, weused previously charaterized monoclonal and polyclonal antibodies assummarized below.
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NHE3 (LL546AP). An affinity-purified polyclonal antibody to NHE3 was previouslycharacterized ( 13, 25 ).
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NaPi-2 (LL697AP/LL696AP). An affinity-purified polyclonal antibody to type NaPi-2 has previouslybeen characterized ( 3 ).
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Na-K-ATPase. A monoclonal antibody against the 1 subunit ofNa-K-ATPase has previously been characterized ( 24 ).& H: ~& Y" R7 q6 ~% T }
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BSC-1 (LL320AP). An affinity-purified polyclonal antibody to the apical Na-K-2Clcotransporter of the thick ascending limb has previously been characterized ( 11, 26 ).; k+ h' t- g5 j
$ _: N& j+ F( u2 ?( CTSC (LL573AP). An affinity-purified polyclonal antibody to the apical Na-Clcotransporter of the distal convoluted tubule has previously beencharacterized ( 27 ).% f0 o7 ?, e/ `5 T
- K1 I; L& l' t W& p! g9 T) CImmunocytochemistry" g( d" ]7 s5 k
! D1 h" v D' g" o% o) zThe kidneys from UUO rats and sham-operated rats were fixed byretrograde perfusion via the abdominal aorta with 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4. For immunoperoxidase microscopy, kidney blocks containing all kidney zones were dehydrated and embeddedin paraffin. The paraffin-embedded tissues were cut (2 µm) on arotary microtome (Leica). The sections were deparaffinated andrehydrated. For immunoperoxidase labeling, endogenous peroxidase wereblocked by 0.5% H 2 O 2 in absolute methanol for10 min at room temperature. To reveal antigens, sections were put in 1 mmol/l Tris solution (pH 9.0) supplemented with 0.5 mM EGTA and heated using a microwave oven for 10 min. Nonspecific binding of Ig was prevented by incubating the sections in 50 mM NH 4 Cl for 30 min, followed by blocking in PBS supplemented with 1% BSA, 0.05%saponin, and 0.2% gelatin. Sections were incubated overnight at 4°Cwith primary antibodies diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. After being rinsed with PBS supplemented with 0.1%BSA, 0.05% saponin, and 0.2% gelatin for 3 × 10 min,the sections were incubated in horseradish peroxidase-conjugatedsecondary antibodies (P448, DAKO) diluted 1:200 in PBS supplementedwith 0.1% BSA and 0.3% Triton X-100, followed by incubation withdiaminobenzidine. The microscopy was carried out using a Leica DMRElight microscope.
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8 O3 T: [$ j/ [% T% GStatistics
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6 l3 r/ h8 e" ^9 uValues are presented as means ± SE. Comparisons betweengroups were made by unpaired t -test. P values significant.
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' ?! Q$ D+ k; e+ hRESULTS
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UUO Impairs Urinary Concentrating Capacity' y1 y2 n" W! _9 E; \& W8 o
9 T C! d; \: C- X) z. A! PPlasma from UUO rats and urine collected from nonobstructedkidneys in the same animals were sampled at the end of the 24-h periodof UUO, and osmolality and concentration of potassium, creatinine,urea, and phosphate were determined (Table 1 ). Twenty-four hours of UUOresulted in a highly significant increase in the concentration ofplasma creatinine (47 ± 1.9 vs. 30 ± 1.1 µmol, P P a parallel increase in plasmaosmolality (308 ± 0.5 vs. 304 ± 1.1 mosmol/kgH 2 O, P 1 ). Urinaryvolume from nonobstructed kidneys was increased significantly duringUUO compared with that in sham-operated control rats (23 ± 2 vs.13 ± 1 µl · min 1 · kg 1;Table 2 ). Furthermore, we examined plasmaand urine samples from both kidneys during a 2-h sampling periodafter release of 24-h UUO for changes in clearance and urinaryexcretion of electrolytes from both the obstructed and nonobstructedkidneys. Compared with sham-operated rats, urine from obstructedkidneys had significantly lower creatinine clearance [0.31 ± 0.16 vs. 1.94 ± 0.24 ml · min 1 · kgbody wt (BW) 1, P 3 ]. Thus glomerular filtration rate(GFR) and urinary concentrating capacity were significantly decreasedin the obstructed kidneys. In contrast, in the nonobstructed kidneys there was a significant increase in urinary volume (7.75 ± 0.91 vs. 3.16 ± 0.26 µl · min 1 · kg 1, P ml · min 1 · kgBW 1, P kidney function (Table 3 ).: j: Z& A9 P1 s9 u6 R
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Table 1. Changes in renal function in rats subjected to 24-h UUO
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Table 2. Changes in renal function in nonobstructed kidneys subjected to 24-hUUO and in right kidneys in sham-operated controls
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Table 3. Changes in renal function in rats subjected to 24-h UUO followed byrelease for 2 h or sham operation
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Excretion of Urinary Sodium, Potassium, and Phosphate Is IncreasedAfter Release of UUO, N3 s5 C/ B3 Y9 K# E' X
6 k* x O5 Z/ x7 ?& y5 A q0 gDuring 24-h UUO, there was no change in plasma and urineconcentrations of sodium and potassium. Plasma phosphate did not change, whereas urinary phosphate increased markedly in UUO rats compared with sham-operated controls (47 ± 6.3 vs. 25 ± 4.8 mmol/l; Table 1 ). The excretion of sodium (3.69 ± 0.44 vs.2.20 ± 0.13 µmol/min), potassium (8.24 ± 0.60 vs.5.19 ± 0.29 µmol/min), and phosphate (0.97 ± 0.09 vs.0.26 ± 0.04 µmol/min) from nonobstructed kidneys increasedmarkedly during 24 h of UUO compared with sham-operated controls(Table 2 ). In addition, fractional excretion of sodium (0.62 ± 0.03 vs. 0.44 ± 0.03%), potassium (47.0 ± 2.1 vs.32.2 ± 2.0%), and phosphate (10.8 ± 0.8 vs. 3.4 ± 0.7%, P nonobstructed kidneys increased(Table 2 ). To examine the effect of UUO on renal sodium handling, ratssubjected to 24 h of UUO were followed for 2 h after releaseof UUO, with urine sampling from both kidneys. Consistent with lowerlevels of creatinine clearance in obstructed kidneys, the filtered loadof sodium and potassium was decreased markedly compared with insham-operated control kidneys and contralateral nonobstructed kidneys.There was a significant increase in urinary sodium excretion (0.29 ± 0.03 vs. 0.17 ± 0.03 µmol/min) and fractional sodiumexcretion (6.4 ± 5.5 vs. 0.7 ± 0.1%, P Urinary excretion of potassium wasmarkedly decreased (0.20 ± 0.06 vs. 0.62 ± 0.08 µmol/min), whereas fractional potassium excretion was increasedmarkedly (158.8 ± 35.7 vs. 70.1 ± 10%, Table 3 ). Increasedurinary sodium and potassium excretion from nonobstructed kidneys wasobserved during 2-h sampling from both kidneys after release of UUO(Table 3 ). These findings show the compensatory increase in theexcretion of sodium, potassium, and phosphate from nonobstructedkidneys during obstruction.% U6 L( p$ s0 l* K
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Decreased Abundance of NHE3 and NaPi-2 in Obstructed Kidneys ofRats With UUO
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- w8 A- O0 H8 T eThe expression levels of major renal sodium transporters wereexamined in both obstructed and nonobstructed kidneys in response to24-h UUO and compared with those in age- and time-matched sham-operated control rats. NHE3 is expressed in renal proximal tubules and TAL and is involved in sodium and bicarbonate reabsorption.Immunoblotting revealed that the abundance of NHE3 was significantlydecreased in the obstructed kidneys of UUO rats (24 ± 4 vs.100 ± 11%, P 2, A and B ), whereas no significantchanges were observed in the nonobstructed kidneys (Fig. 2, C and D ).
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4 y! E# @* Z' s/ ?' uFig. 2. Semiquantitative immunoblotting of membrane fractions of outermedulla and cortex from UUO and sham-operated rats. A and C : immunoblots were reacted with affinity-purifiedanti-type 3 Na/H exchanger (NHE3) antibody and revealed a single~87-kDa band. OBS, obstructed. B : densitometric analysis(corrected according to densitometry of Coomassie-stained gels andloading fraction of total kidney mass; see METHODS ) of allsamples from obstructed kidneys (OBS) of rats with 24-h UUO andsham-operated controls revealed a significant decrease in NHE3 levelsfrom 100 ± 11% in sham-operated controls to 24 ± 4% inobstructed kidneys. D : in nonobstructed kidneys (non-OBS),densitometric analysis revealed no difference from sham controls(81 ± 14 vs. 100 ± 10%).: o5 a4 ~% a3 X- l
" h& e( d6 B, \# _' J2 cImmunocytochemical analysis confirmed these results. In sham-operatedrats, anti-NHE3 antibody labeled the apical plasma membrane domains ofproximal convoluted tubules (Fig. 3 E ), as previously described( 1 ). Furthermore, intense labeling of the apical plasmamembrane domains of mTAL cells in sham-operated rats was also seen(Fig. 3 F ). In contrast, immunocytochemistry showed that thelabeling of NHE3 in the proximal tubule cells as well as of TAL cellsfrom the obstructed kidneys was reduced (Fig. 3, A and B ). The labeling density did not change in the nonobstructedkidneys (Fig. 3, C and D ), consistent with theimmunoblotting data. These findings indicate that reduced abundance ofNHE3 in the obstructed kidney may contribute to increased sodiumexcretion observed in response to release of UUO.3 \& \' @0 L4 Y# @- u' N k
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Fig. 3. Immunocytochemical analyses of NHE3 in proximal tubules and thickascending limb (TAL) of the loop of Henle from sham-operated and UUOrats (obstructed and nonobstructed kidney). A and B : in the obstructed kidneys of UUO rats, NHE3 labeling (arrows)is seen at apical domains of proximal tubule cells ( A ) andat apical membrane domains in TAL cells ( B ), but labeling ismuch weaker compared with that seen in kidneys from sham-operated rats. C and D : in the nonobstructed kidneys of UUO rats, NHE3labeling is comparable to that seen in sham-operated controls. E : in sham-operated rats, NHE3 labeling is seen at apicaldomains of proximal tubule cells but is absent in brush-border andbasolateral membrane domains. F : in sham-operated rats, NHE3labeling is seen at apical membrane domains of the thick ascending limbin the inner stripe of the outer medulla (ISOM). P, proximal tubule; T,thick ascending limb; D, distal convoluted tubule; CD, collecting duct.Magnification: ×650.
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0 x9 `" r' k9 `9 d$ `2 sIn the apical part of the renal proximal tubules, NaPi-2 contributes tosodium and phosphate reabsorption. As shown in Fig. 4, immunoblotting revealed a markeddecrease in NaPi-2 levels in both obstructed and nonobstructed kidneys(21 ± 4 vs. 22 ± 4% of control levels, P NaPi-2 may also contribute tothe increased urinary sodium excretion and is also likely to play amajor role in increased urinary phosphate excretion.
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7 U& R9 c. H9 o% LFig. 4. Immunoblot of membrane fractions of outer medulla and cortex fromUUO and sham-operated rats. A and C : immunoblotswere reacted with affinity-purified anti-type 2 Na-P i cotransporter (NaPi-2) antibody and revealed a single ~85-kDa band. B : densitometric analysis of all samples from obstructedkidneys of rats with 24-h UUO and sham-operated controls revealed amarked decrease in NaPi-2 levels in obstructed kidneys to 21 ± 4% compared with sham-operated rats (100 ± 12%,* P D : densitometric analysis ofall samples from nonobstructed kidneys of rats with 24-h UUO andsham-operated controls also revealed a marked decrease in NaPi-2 levelsin nonobstructed kidneys to 22 ± 4% compared with sham-operatedcontrols (100 ± 27%, * P
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Abundance of Na-K-ATPase Is Reduced in Obstructed Kidneys of RatsWith UUO. B! X' ^3 B) E
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Immunoblotting of the -subunit of the Na-K-ATPaseshows that the abundance is markedly decreased in the outermedulla and cortex of obstructed kidneys (37 ± 4 vs. 100 ± 6%, P 5,Table 4 ), whereas the abundance isunchanged in the inner medulla (Fig. 6,Table 4 ). In contrast, in nonobstructed kidneys the abundance in theinner medulla was significantly reduced (46 ± 10 vs. 100 ± 12%, P 6, Table 5 ), whereas there was no change in theouter medulla and cortex (90 ± 12 vs. 100 ± 15%, notsignificant; Fig. 5, Table 5 ).
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) z3 u7 W0 |0 m3 zFig. 5. Semiquantitative immunoblotting of membrane fractions of outermedulla and cortex (OM C) from UUO and sham-operated rats. A andC : immunoblots were reacted with -isoform-specific Na-K-ATPase. B : densitometric analysis of all samples from the obstructedkidneys of rats with 24-h UUO and sham-operated controls revealed amarked decrease in expression from 100 ± 6% in sham-operatedcontrols to 37 ± 4% in obstructed kidneys (* P D : densitometric analysis revealed that Na-K-ATPaseexpression (90 ± 12%) in nonobstructed kidneys did not differfrom levels in sham-operated controls (100 ± 15%).
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Table 4. Expression of sodium cotransporters in obstructed kidneys of rats with24-h UUO and sham-operated controls
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$ ?$ i- Z. `% D' LFig. 6. Semiquantitative immunoblotting of membrane fractions of innermedulla (IM) from UUO and sham-operated rats. A and C : immunoblots were reacted with monoclonal antibody specific tothe -subunit of Na-K- ATPase and revealed a single ~96-kDa band. B : densitometric analysis of all samples from obstructedkidneys of rats with 24-h UUO and sham-operated controls revealed nodifference between obstructed kidneys and controls (94 ± 16 vs.100 ± 16%). D : in nonobstructed kidneys,densitometric analysis revealed a significant reduction (to 46 ± 10%) compared with controls (100 ± 12%, * P
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7 s+ s8 W# d' E& ]; |# QTable 5. Expression of sodium cotransporters in nonobstructed kidneys in ratswith 24-h UUO and sham-operated controls5 z& q9 i7 T* B6 {
1 B$ Y# s- Z9 X# @; _- [/ | EAbundance of BSC-1 and TSC Is Reduced in Obstructed Kidneys of RatsWith UUO' T9 j5 J; K$ t* T3 ~6 A$ ]
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The abundance of BSC-1 in the outer medulla and cortex wasdramatically decreased (15 ± 3 vs. 100 ± 13%, P 7, A and B ). In contrast, BSC-1abundance was unchanged in nonobstructed kidneys (Figs. 7, C and D ) compared with sham-operated rats. Immunocytochemistry showed that the labeling of BSC-1 in the mTAL of obstructed kidneys wasmarkedly decreased (Fig. 8 A ). In nonobstructed kidneys,labeling of BSC-1 in the mTAL was unchanged compared with that insham-operated rats (Fig. 8, B and C )./ R8 W/ |0 [3 \9 ]
+ T+ x1 \$ {$ GFig. 7. Semiquantitative immunoblotting of membrane fractions of outermedulla and cortex from UUO and sham-operated rats. A and C : immunoblots were reacted with affinity-purified type 1 bumetanide-sensitive Na-K-2Cl contransporter (BSC-1) antibody andrevealed a strong, broad band of 146- to 176-kDa molecular masscentered at ~161 kDa. B : densitometric analysis of allsamples from obstructed kidneys of rats with 24-h UUO and sham-operatedcontrols revealed a marked decrease in obstructed kidneys (15 ± 3%) compared with control rats (* P D : densitometric analysis revealed that BSC-1 abundance wasunchanged in nonobstructed kidneys compared with sham-operatedcontrols.# x# x3 X0 f4 w" {0 G
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Fig. 8. Immunocytochemical analyses of BSC-1 in the medullary(m)TAL of UUO rats (obstructed and nonobstructed kidney) andsham-operated rats. A : in the obstructed kidneys of UUOrats, labeling of BSC-1 (arrows) in TAL cells is much weaker comparedwith in sham-operated kidneys ( C ). B : in thenonobstructed kidneys of UUO rats, labeling of BSC-1 in TAL cells iscomparable to that seen in kidneys of sham-operated controls. C : abundant BSC-1 labeling is seen in apical plasma membranedomains of mTAL cells in inner stripe of outer medulla fromsham-operated controls. Magnification: ×650.
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$ Z2 L) B2 t! Y, a+ b; L7 lTSC abundance was markedly reduced in obstructed (15 ± 4 vs.100 ± 8%, P 9, A and B ) but not in nonobstructed kidneys (Fig. 9, C and D ). Consistent with this,immunocytochemistry revealed that labeling of TSC seen at the apicalmembrane domains of DCT cells was markedly reduced in obstructedkidneys (Fig. 10 A ), whereasno change in labeling was found in nonobstructed kidneys (Fig. 10 B ). These results suggest that BSC-1 and TSC may also playsignificant roles in the changes in sodium and water handling inkidneys with urinary tract obstruction., `/ H2 F5 \4 z2 `# i# \
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Fig. 9. Semiquantitative immunoblotting of membrane fractions of outermedulla and cortex from UUO and sham-operated rats. A and C : immunoblots were reacted withaffinity-purified anti-thiazide-sensitive Na-Cl cotransporter (TSC)antibody and revealed a broad band centered at ~165-kDa. B : densitometric analysis of all samples from obstructedkidneys of rats with 24-h UUO and sham-operated controls revealed amarked decrease in TSC expression to 15 ± 4% in obstructedkidneys compared with sham-operated controls. * P D : densitometric analysis revealed that TSC abundancewas unchanged in nonobstructed kidneys compared with sham-operatedcontrols.0 H9 k; D Q+ u& m
/ ~* G, \3 C% `! C1 l% FFig. 10. Immunocytochemical analyses of TSC in DCT ofsham-operated and UUO rats (obstructed and nonobstructed kidney). A : in the obstructed kidneys of UUO rats, labeling of TSC(arrows) in DCT cells is much weaker compared with sham-operatedkidneys ( C ). B : in the nonobstructed kidneys ofUUO rats, labeling of TSC in DCT cells is similar to the labeling seenin sham-operated controls. C : TSC labeling is seen at apicalplasma membrane domains of DCT cells in sham-operated rats.Magnification: ×1,100.
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This study demonstrated that UUO is associated with an impairedurinary concentrating capacity and salt wasting in the obstructed kidney. In parallel, the expression of several major renal sodium transporters, i.e., NHE3, NaPi-2, Na-K-ATPase, BSC-1, and TSC, weresignificantly downregulated in the obstructed kidney, suggesting thatreductions in these major renal sodium transporters contribute to theimpaired urinary concentrating capacity and salt wasting after releaseof UUO. In the contralateral nonobstructed kidney, immunoblottingrevealed a reduced expression of Na-K-ATPase in the inner medulla and areduced expression of NaPi-2 in the proximal tubule. This wasassociated with an increased excretion of sodium and phosphate duringand after release of UUO. These findings demonstrate segmental changesat the molecular level in sodium and phosphate transporters in both theobstructed and nonobstructed kidneys during UUO., j' ?, E1 m; |1 ~7 g- J' x
; h# L" ~( `1 d* }7 ^& IUUO Is Associated With Salt Wasting
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Urinary tract obstruction results in profound changes in renalhemodynamics. The present study showed that UUO resulted in a dramaticreduction in GFR in the obstructed kidney when creatinine clearance wasmeasured 2 h after release, consistent with previous studiesdemonstrating a marked GFR reduction due to severe vasoconstriction ( 17, 20 ). This reduction in GFR in the postobstructedkidney reduces the filtered load of sodium, which is consistent with the finding of the present study that the filtered load of sodium wasseverely reduced in the obstructed kidney. During UUO, these hemodynamic changes are associated with disruption of tubule function, which ultimately results in salt wasting and an impaired urinary concentrating capacity of the obstructed kidney ( 6 ).Despite the reduced load of sodium entering the tubules, UUO wasassociated with a significant increase in urinary sodium excretion.Thus sodium reabsorption was severely impaired during the passageof the ultrafiltrate through the tubules and collection ducts.Previous studies have indicated that the major defects in renaltubular sodium reabsorption are located in the distal segments of the nephron. Micropuncture studies have shown a diminished netreabsorption of both sodium and water between the loop of Henle andthe beginning of the papillary CD ( 41, 48, 50 ),demonstrating that these segments of the nephron are criticallyaffected by ureteral obstruction. The results of the present studytogether with previous findings (Li, Wang, Knepper, Nielsen, andFrøkiær, unpublished observations) support the view that the distalsegments of the nephron and collecting ducts play a significantrole in the impairment in urinary concentrating capacity of theobstructed kidney.- j5 @1 v0 P. _8 J
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In the contralateral nonobstructed kidney, GFR increased during 24 h of UUO. This resulted in an increased filtered load of sodium and adramatic increase in urinary sodium excretion from the nonobstructedkidney. Importantly, analysis of urine collected during UUO from thecontralateral nonobstructed kidney demonstrated an increased excretionof both sodium and phosphate. Thus UUO is associated with sodium andphosphate loss caused by dysregulation of sodium transporters in boththe obstructed and contralateral nonobstructed kidney.
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Reduced Abundance of Sodium Transporters in the Proximal Tubule inUUO
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' s+ X5 o/ z, n& t8 J( [The present study demonstrated that the abundance of NHE3 andNaPi-2 in the obstructed kidney were severely decreased 24 h afterthe onset of obstruction. Downregulation of NHE3 in the proximal tubulewas confirmed by immunocytochemistry. The observation that theaforementioned two major proximal sodium transporters are reduced inthe obstructed kidney supports the view that the epithelial transportability of the proximal tubule is impaired in the obstructed kidney inresponse to UUO. Furthermore, the abundance of Na-K-ATPase in thecortex and outer medulla of the obstructed kidney was reduced after24-h UUO. This finding is consistent with previous studiesdemonstrating markedly reduced Na-K-ATPase activity in basolateralmembrane vesicles prepared from the cortex of the obstructed kidneys ofrats with 24-h UUO ( 5 ).
4 Y, E& m ~3 B9 ~
9 K; b' L* k. F- p/ QIt is evident that functional adaptation of the proximal tubule mayplay a critical role in the changes in reabsorption and secretion ofsodium. In vitro experiments have previously demonstrated that volumereabsorption in proximal straight tubules was diminished in obstruction( 19 ). Regulated apical membrane Na/H exchange is a majorpathway for proximal tubule sodium reabsorption. NHE3 has been shown tobe responsible for this ( 1 ). The reduced abundance ofNaPi-2 in the obstructed kidney may contribute to the impairment ofsodium reabsorption at the proximal tubule. Furthermore, downregulationof NaPi-2 may affect renal proximal tubular handling of phosphate inthe obstructed kidney. Consistent with the increased urinary excretionand fractional excretion of sodium, UUO was associated with a markedreduction in proximal tubular sodium transporter abundance (i.e., NHE3,NaPi-2, and Na-K-ATPase). Thus it is likely that the reduction inexpression of these proximal tubule sodium transporters plays asignificant role in the increased urinary sodium excretion after UUO.During UUO, renal function of the obstructed kidney is graduallyimpaired due to severe vasoconstriction, which leads to reduced bloodsupply; the obstructed kidney then becomes gradually ischemicin response to obstruction ( 28 ). It may be ofinterest to note that the reduction in proximal tubular sodiumtransporters in the obstructed kidney (NHE3, NaPi-2, andNa-K- ATPase) is similar to that demonstrated in response to acuteischemia-induced renal failure ( 29 ). The presentfindings demonstrate that 1 ) the proximal tubule issusceptible to injury in response to ureteral obstruction, consistentwith previous observations ( 19 ); and 2 )reabsorption of sodium and phosphate at the proximal tubular level maybe severely compromised due to the significant reduction in theexpression of proximal tubular sodium transporters.
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) R9 r+ |- P4 x5 q4 fReduced Abundance of Sodium Transporters in the TAL and DCT DuringUUO8 ]9 h% G8 |$ f+ |
- v$ T9 m' c, Z3 N) U8 N# Z2 k% kThe mTAL of Henle is known to be susceptible toischemia-induced injury ( 28 ). Thus the progressiveischemia induced by ureteral obstruction may change thefunction of the mTAL. BSC-1, which is localized at the apical plasmamembrane domains of mTAL and cortical TAL (cTAL) segments ( 11, 26 ), and the basolaterally located Na-K-ATPase ( 24 )are the key sodium transporters that mediate the NaCl transport inthese water-impermeable segments. The countercurrent multiplicationprocess is dependent on the active reabsorption of NaCl in mTAL( 7, 38 ). Thus these pathways are critical for thegeneration of the hypertonic medullary interstitium. In particular, theapically expressed Na-K-2Cl cotransporter in TAL is known to beregulated by vasopressin ( 26 ), and this regulation may beinvolved in the long-term regulation of the countercurrent multiplication system ( 7 ). Importantly, the present studydemonstrated both by immunoblotting and by immunocytochemistry that theabundance of BSC-1 and Na-K-ATPase was significantly decreased inobstructed kidneys of rats with UUO. This finding is consistent withprevious studies indirectly demonstrating reduced activities of boththe Na-K-2Cl cotransporter and Na-K-ATPase by reductions in theinhibitory effects of both furosemide and ouabain in suspensions ofmTAL cells from obstructed kidneys ( 21 ). This findingstrongly indicates that the reduced expression of BSC-1 plays a keyrole in the reduced reabsorption of sodium and chloride in the TAL inresponse to UUO. Moreover, the reduced expression is likely tocontribute to the decreased urinary concentration and increased urinarysodium excretion from the obstructed kidney.; P- Q9 M5 Y3 Y
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Several studies have demonstrated that vasopressin increases the rateof net sodium reabsorption in microperfused TAL segments from mice andrats ( 18, 40 ) and that the abundance of Na-K-2Cl cotransporter in the TAL is increased in response to dDAVP( 26 ). Vasopressin administration also stimulates cAMPproduction. However, in the postobstructed kidney vasopressinadministration does not stimulate cAMP ( 28 ), whichsupports the view that the impaired sodium reabsorption in the TAL iscaused in part by a reduced vasopressin sensitivity in the obstructedkidney. In contrast, PGE 2 decreases the rate ofvasopressin-stimulated NaCl reabsorption in TAL ( 8, 14, 42 ). Previously, it was demonstrated that the accumulation ofvolume and osmotic substances during obstruction may stimulate renalsecretion of PGE 2 ( 35 ), which may have an inhibitory effect on the TAL cAMP levels, and hence this may influence the abundance of Na-K-2Cl cotransporter. Therefore, it is possible thatboth the impaired sensitivity to vasopressin and potential PGE 2 -induced cAMP inhibition may contribute to thereduction in BSC-1 abundance in the obstructed kidney after UUO.
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In the DCT, TSC is expressed and responsible for a large proportion ofthe apical sodium reabsorption that takes place in this segment of thenephron ( 27, 34 ). In the present study, we demonstratedthat the abundance of TSC was severely reduced in the obstructedkidney. This was confirmed by immunocytochemistry, demonstrating muchweaker labeling at the apical membrane of the DCT. The abundance of TSCprotein appears to be highly regulated. TSC abundance in the rat kidneyis increased in response to aldosterone infusion or use of a low-saltdiet ( 27 ), by nitric oxide synthase inhibition( 44 ), by estrogen infusion ( 45 ), and duringvasopressin escape ( 9 ). The renin-angiotensin-aldosteronesystem plays a pivotal role in the pathophysiology of obstructivenephropathy. Although we did not measure aldosterone levels in thepresent study, 24-h UUO has been demonstrated to increase plasmaaldosterone levels ( 12 ). Consequently, the downregulationof TSC in response to obstruction may be speculated to be caused by aninsensitivity to the mineralocorticoid receptor or due to disruption ofintracellular pathways regulating TSC expression. The discovery thatboth BSC1 and TSC are reduced in the obstructed kidney demonstratesthat 1 ) the TAL and DCT are highly susceptible to injuryinduced by obstruction; and 2 ) sodium reabsorption may beseverely compromised, which contributes to the impairment of theurinary concentrating capacity and to increased urinary sodium loss.- `+ k' d" p; M" L9 r) g6 A/ \# @
& m1 R4 U" I0 i: M3 W* HThe demonstration of reduced abundance of sodium transporters in bothproximal and distal nephron segments suggests that the mechanismresponsible for the downregulation is complex and cannot be ascribed toa single mediator. The increased pressure associated with 24 h ofUUO may be of crucial importance, because it is well known thatnumerous hormonal and inflammatory pathways are activated in responseto UUO ( 28 ).* ?0 R r: s1 z! ]2 i- [
, `% F. |( [0 j: j7 o3 T
Abundance of Sodium Transporters in the Contralateral NonobstructedKidney6 m9 ~# z& o% S
3 M! J, M2 Y. h" z7 F% SIt is well known that during UUO the contralateral kidneyundergoes adaptational changes due to the loss of function of the obstructed kidney. In the present study, there was a dramatic reductionin the abundance of NaPi-2 in the cortex and outer medulla and amoderate reduction in the abundance of Na-K-ATPase in the inner medullaof nonobstructed kidneys. Consistent with these findings, the excretionof both phosphate and sodium was markedly increased from nonobstructedkidneys during UUO. This is a new finding, demonstrating changes at themolecular level in both the proximal tubule and the collecting ducts ofthe contralateral nonobstructed kidney, indicating that regulation ofsodium transporters in these segments plays an important role in thecompensatory response to UUO. It is well known that the intactkidney compensates for the reduction in renal function in response touninephrectomy and UUO ( 28 ). However, the present resultsare the first to demonstrate compensation of renal sodium transport atthe molecular level in response to UUO. Additional studies inresponse to uninephrectomy are required to identify whether thesechanges are of general significance as a compensatory response tounilateral kidney disease.
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3 Z4 c2 o; h% w- u. ?UUO in rats is associated with significant reductions in theabundance of major renal sodium transporters along the nephron. Importantly, the abundance of both apically and basolaterally locatedsodium transporters was reduced, consistent with a significant impairment of tubular reabsorption of filtered sodium. We conclude that 1 ) downregulation of major renal sodium transporters in rats with UUO may contribute to the impairment in urinary concentrating capacity and natriuresis after release of obstruction and 2 )reduced levels of NaPi-2 and Na-K-ATPase in the contralateralnonobstructed kidney may contribute to the compensatory increase inwater and sodium excretion from that kidney during UUO.
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ACKNOWLEDGEMENTS* h8 g8 G( S, p' G
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The authors thank Gitte Christensen, Dorte Wulff, Inger MeretePaulsen, Mette Vistisen, Helle Høyer, Zhile Nikrozi, Lotte ValentinHolbech, and Merete Pedersen for technical assistance.
! B1 h% Y0 T0 |" D8 t2 G 【参考文献】
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9 s" t) i& j; ], i28. Klahr, S. Obstructive nephropathy.In: Textbook of Nephrology, edited by Massry SG,and Glassock RJ.. Philadelphia, PA: Lippincott Williams & Wilkins, 2001, p. 987-1003.
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