Renal expression of sodium transporters and aquaporin-2 inhypothyroid rats - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:36 |只看该作者 |倒序浏览 |打印

作者：RolandSchmitt, EnnoKlussmann, ThomasKahl, David H.Ellison,  SebastianBachmann作者单位：1 Institut für Anatomie, Charité, HumboldtUniversität, 10115 Berlin; Forschungsinstitutfür Molekulare Pharmakologie, Campus Berlin-Buch, 13125 Berlin,Germany; and Division of Nephrology, Hypertension,and Clinical Pharmacology, Oregon Health Sciences University,Portland, Oregon  * s- J$ A4 F3 k4 g% C) n* n6 O! y

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      【摘要】
   Hypothyroidism is associatedwith significant abnormalities in the renal handling of salt and water.To address the involvement of tubular transport proteins in theseabnormalities, rats were rendered pharmacologically hypothyroid and theabundance of major tubular transport proteins was assessed byimmunoblot and immunohistochemistry. Hypothyroidism resulted in amarked reduction in kidney size and creatinine clearance along withdecreased or unchanged total kidney abundance of the transportproteins. Whereas the proximal tubular type 3 Na/H exchanger (NHE3) andtype 2 Na-phosphate cotransporter (NaPi2) stood out by theirdisproportionately reduced abundance, the bumetanide-sensitive type 2 Na-K-2Cl cotransporter (NKCC2) and aquaporin-2 (AQP2) were unaltered intheir total kidney abundance despite a markedly lower kidney mass. Thelatter proteins in fact showed enhanced immunostaining. Decreased NHE3and NaPi2 expression was most likely due to a combination oftriiodo- L -thyronine (T 3 ) deficiency along witha reduced glomerular filtration rate. The increased abundanceof NKCC2 and AQP2 may have been caused by an increased action ofvasopressin since urinary excretion of this hormone was elevated. Onthe other hand, the thiazide-sensitive Na-Cl cotransporter; the -, -, and -subunits of the amiloride-sensitive epithelial Nachannel; and the 1 -subunit of Na-K-ATPase showed amoderate decrease in total kidney abundance that was largely proportional to the smaller kidney mass. Although the observed expression of transporters was associated with a balanced renal sodiumhandling, altered transporter abundance may become functionally relevant if the hypothyroid kidney is challenged by an additional destabilization of the milieu interieur that has previously been shownto result in an inadequate natriuresis and clinical symptoms.
      【关键词】 sodium transport vasopressin distal tubule* @1 o, q  S; u
   INTRODUCTION! S) v3 W) M: a4 g
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HYPOTHYROIDISM IS ASSOCIATED with significant abnormalities of renal function inhumans and in experimental animals. The impairment of renal functiontypically manifests as a reduction in glomerular filtration rate (GFR),reduced diluting and concentrating ability, and exaggerated natriuresisin response to volume alterations caused by saline and water loading orother (for a review, see Refs. 7, 20, 25 ). The latter deficiency has been so marked that ratsreceiving antithyroid drugs together with a sodium-deficient diet wererapidly dying from negative sodium balance ( 16 ). The observed sodium loss was attributed to a diminished reabsorptive ability of the proximal ( 31 ) and the distal renal tubule( 15, 21 ) as revealed by micropuncture studies. One of themechanisms resulting in the reabsorptive defect was suggested to be thedependency of tubular sodium reabsorption on the activity ofNa-K-ATPase, which is known to be reduced in hypothyroid rats( 22 ). However, it has been demonstrated that the reducedacitivity of Na-K-ATPase alone cannot account for the transport changesobserved in hypothyroidism ( 10, 11 ). Although Na-K-ATPaseactively maintains ionic gradients, it is the passive apical entrythrough various transport proteins that normally represents therate-limiting step in tubular sodium reabsorption. Thus a potentialmechanism accounting for the insufficient reabsorption of thehypothyroid kidney may be the decrease in the abundance or activity ofthe apical sodium transport pathways. The influence of thyroid hormoneon two major sodium transporters of the proximal tubule, the type 3 Na/H exchanger (NHE3) and the type 2 Na-phosphate cotransporter(NaPi2), has recently been investigated and it has been shown that bothproteins are directly regulated by thyroid hormone ( 1, 9, 41 ). On the other hand, no information is available on theexpression of the major distal tubular sodium transporters, i.e., thebumetanide-sensitive type 2 Na-K-2Cl cotransporter (NKCC2) of the thickascending limb of the loop of Henle (TAL), the thiazide-sensitive Na-Clcotransporter (NCC) of the distal convoluted tubule (DCT), and theamiloride-sensitive epithelial Na channel (ENaC) of the late DCT,connecting tubule (CNT), and the cortical and medullary collecting duct(CCD, MCD), in altered thyroid status. We studied the hypothesis ofwhether renal expression of these proteins would be altered in ratspharmacologically rendered hypothyroid to elucidate their possibleimplication in impaired sodium handling. To this end, abundance ofthese proteins was analyzed by semiquantitative immunoblotting andimmunohistochemistry. Further key sodium transporters such as theproximal tubular NHE3 and NaPi2 as well as the 1 -subunitof Na-K-ATPase were also analyzed. To address water transport in thelate distal tubule and collecting duct, we extended our observations tothe expression of the water channel aquaporin-2 (AQP2), which mediateswater permeability across the luminal membrane of these segments( 27, 34 ) and may therefore be critical in anotherprominent feature of hypothyroidism, i.e., the impairment in waterreabsorption ( 38 ).
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MATERIALS AND METHODS
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Animal model. Sixteen male Sprague-Dawley rats weighing 150 g (Schönwalde,Berlin, Germany) were divided randomly into two groups. Both groupswere fed the same standard diet and had free access to drinkingwater. Hypothyroidism was induced by adding 0.05% methimazole to thewater in one group. Body weights were measured weekly. After 7 wk oftreatment, urine was collected for 20 h by placing the rats intoindividual metabolic cages. Thereafter, rats ( n = 6)were in vivo perfusion-fixed for immunohistochemistry. For biochemicalanalysis of serum and kidneys, rats ( n = 10) were killed by an overdose of pentobarbital sodium; blood was collected bypuncture of the abdominal aorta, and both kidneys were quickly removed,individually weighed, decapsulated, cut into small pieces, frozen inliquid nitrogen, and stored at 80°C. To exclude a direct effect ofmethimazole on transporter abundance, an additional group of rats( n = 5) receiving 0.05% methimazole during 4 wk wassubstituted with triiodo- L -thyronine (T 3 ) by asubcutaneous implantation of 0.05 mg T 3 -releasing pellets[IRA, Sarasota, FL ( 1 )]; pellets were implanted in thesecond week to deliver T 3 during the following 3 wk.

Tissue preparation for immunoblotting. The tissue was homogenized for 3 min using a tissue homogenizer (Diax600, Heidolph Instruments) in an ice-cold solution containing 250 mMsucrose, 10 mM triethanolamine with 1 tablet/50 ml of a proteaseinhibitor cocktail (Complete, Roche Diagnostics, Mannheim, Germany).The whole kidney homogenate was centrifuged at 1,000 g for10 min to remove nuclei and incompletely homogenized membrane fragments. The supernatant was then centrifuged at 200,000 g for 1 h to obtain a pellet containing plasma membranes andintracellular vesicles. The 1,000- g centrifugation wascarried out using a Sigma 3K15 refrigerated centrifuge. The200,000- g spin was carried out with a Beckman LE 80 ultracentrifuge. The resulting pellets were resuspended inhomogenization solution, and total protein concentration was measuredusing the Pierce BCA Protein Assay reagent kit (Pierce, Rockford, IL).

Electrophoresis and immunoblotting of membrane proteins. After Laemmli's sample buffer was added, the proteins were solubilizedat 65°C for 10 min. SDS gel electrophoresis was performed on6-10% polyacrylamide gels. After electrophoretic transfer of theproteins to polyvinylidene fluoride membranes, equity in protein loading and blotting was verified by membrane staining using 0.1% Ponceau red. Membranes were probed overnight at 4°C or atroom temperature for 2 h and then exposed to horseradishperoxidase-conjugated secondary antibodies (DAKO, Glostrup, Denmark)for 45 min at room temperature. Immunoreactive bands were detected onthe basis of chemiluminescence, using an enhanced chemiluminescence kit(Amersham Pharmacia, Freiburg, Germany) before exposure to X-ray films(Hyperfilm, Amersham). For densitometric evaluation of the resultingbands, films were scanned and analyzed using BIO-PROFIL Bio-1D image software (Vilber Lourmat, Marue La Vallee, France).$ d' ]& D% H6 P1 g6 f9 t
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Antibodies. We used previously well-characterized rabbit polyclonal antibodies tothe following proteins: NaPi2 [antibody was a gift from J. Biber,Department of Physiology, University Zürich ( 43 )], NCC ( 39 ), AQP2 ( 28 ), and -ENaC( 39 ). For - and -ENaC, commercially availablepolyclonal rabbit antibodies were used (Alpha Diagnostic International,San Antonio, TX). Mouse monoclonal antibody against NHE3 was purchasedfrom Chemicon International (Temecula, CA), and monoclonal antibodyagainst the 1 -subunit of Na-K-ATPase was from UpstateBiotechnology (Lake Placid, NY). Polyclonal antibodies against NKCC2were raised in guinea pigs against a rat NH 2 -terminalNKCC2-maltose-binding fusion protein (MBP), which was produced using aDNA fragment encoding the 85 NH 2 -terminal amino acids ofNKCC2. The fragment was cloned inframe into pMAL (New England BioLabs,Beverly, MA), and the construct was verified by sequencing. NKCC2-MBPwas produced and then purified using an amylose affinity column.One hundred micrograms of NKCC2-MBP together with Freund's adjuvantwere used for immunization.
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Tissue preparation for immunohistochemistry. The animals were killed by in vivo perfusion fixation underpentobarbital sodium anesthesia. The kidneys were perfused retrograde through the abdominal aorta using PBS adjusted to 330 mosmol/kgH 2 O with sucrose, pH 7.4, for 20 s. Next, 3%paraformaldehyde in PBS was infused for 5 min, followed by the firstsolution for an additional minute. The kidneys were removed and cutinto slices. Slices were processed for embedding in Epon and paraffin,or they were shock-frozen in liquid nitrogen-cooled isopentane forsubsequent cryostat sectioning.% w' a0 \, g# j- c, R
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Immunohistochemistry. Immunohistochemical staining was performed on cryostat sections or onsections from Epon- and paraffin-embedded tissue. For labeling of NCC,0.5-µm Epon sections were pretreated with 5-min steps of Namethanolate, methanol-toluol, and acetone before blocking with 5%skimmed milk in PBS. For detection of -, -, -EnaC, and 1 -Na-K-ATPase, 4-µm cryostat sections were cut andmilk-blocked. After the sections were incubated with the respectiveprimary antibody at 4°C overnight, bound antibody was detected by a1-h incubation with Cy3-conjugated goat anti-rabbit or goat anti-mouse antibody (DIANOVA, Hamburg, Germany). For immunolabeling of NKCC2 andAQP2, dewaxing and rehydration of 4-µm paraffin sections were followed by 10 min in 0.5% H 2 O 2 in methanol toblock endogenous peroxidase. Improved antigen retrieval was achieved byheating the sections in 0.01 mM Na citrate using a microwave oven atmaximal power for 15 min. After being blocked with 5% skimmed milk in PBS, sections were incubated overnight at 4°C with primaryantibodies. Antibody detection was carried out using horseradishperoxidase-conjugated rabbit anti-guinea pig antibodies (DAKO) forNKCC2 or swine anti-rabbit IgG (DAKO) followed by rabbitanti-horseradish peroxidase IgG (DAKO) for AQP2. Generation of signalwas realized with 0.1% diaminobenzidine and 0.02%H 2 O 2 in PBS for standardized duration. Sectionswere counterstained with hematoxylin and analyzed using a Leica DMRB microscope.

Blood and urine analysis. Thyroid hormones were measured by a chemiluminescent immunoassay(Centaur analyzer, Bayer, Germany) in plasma samples collected asdescribed above. Electrolytes in serum and urine were determined byindirect ion-selective electrode analysis (Modular Analytics, RocheDiagnostics), and urinary osmolality was measured by an osmometer(Gonotec, Berlin, Germany). Blood Urea Nitrogen (BUN) and creatinineconcentrations were determined enzymatically and by the kineticJaffé-method (Modular Analytics, Roche Diagnostics). Creatinine clearance and fractional sodium excretion were calculated using standard equations. Measurement of urinary vasopressin in 20-hurine collections was kindly performed by S. Diederich, Free UniversityBerlin, using a radioimmunoassay as previously described ( 12 ).
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Presentation of data and statistical analysis. Quantitative data are presented as means ± SE. For statisticalcomparison, the unpaired t -test (when variances were thesame) or the Mann-Whitney rank sum test (when variances differedsignificantly between groups) was employed. P values of lessthan 0.05 were considered statistically significant., H/ P. c' s6 V
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RESULTS! N$ m9 R' W  c1 {

Clinical parameters and functional data. As shown in Table 1, treatment withmethimazole resulted in dramatically decreased serum levels of freeT 3 and thyroxine (T 4 ) and was associated with asignificant lack of weight gain (mean body wt 179 ± 6 vs. control358 ± 8 g). In addition to the lower body weight, kidneysshowed a disproportionately reduced weight, resulting in a loweredkidney-to-body weight ratio (0.0046 ± 0.02 vs. control0.0055 ± 0.02). Analysis of renal function revealed asignificantly increased 20-h urinary output (15.7 ± 0.9 vs.control 9.4 ± 1 ml), which was paralleled by a marked reductionin urinary osmolality (534 ± 43 vs. control 1,805 ± 308 mosmol/kgH 2 O). The creatinine clearance was significantly reduced (0.7 ± 0.09 vs. control 1.64 ± 0.5 ml · min 1 · g kidney wt 1 ),whereas the fractional Na excretion (FE Na ) was markedlyincreased (0.69 ± 0.3 vs. control 0.2 ± 0.12%). There wereno significant changes in serum Na (143.4 ± 0.7 vs. control142.2 ± 2.1 mmol/l), but there was a significant elevation inserum levels of creatinine and urea nitrogen (0.61 ± 0.05 vs.control 0.32 ± 0.05 mg/dl and 95.2 ± 7.9 vs. control46.8 ± 8.2 mg/dl). A possible role of an altered vasopressinstatus was studied by measuring the hormone in 20-h urine collections,since urinary vasopressin concentrations are known to indicate changesin plasma vasopressin concentrations ( 12 ). Urinaryconcentrations of vasopressin were not different between hypothyroidsand controls (70.1 ± 23 vs. 72.02 ± 12 pg/ml). Related tothe highly divergent urinary volumes between groups, however, asignificantly higher vasopressin excretion was found in the hypothyroidrats (1,084 ± 203 vs. 678 ± 119 pg/20 h).

Table 1. Clinical parameters and functional data on renal function- O% i2 ^! S7 n; N( p

Histology and abundance/distribution of tubular transporters andchannnels. General histology revealed a marked reduction in size of the glomeruli,and tubules were also significantly smaller in profile in hypothyroidanimals than in controls. Apart from these changes, there were nofurther obvious alterations in renal structure.

NHE3 and NaPi2. Antibodies against NHE3 and NaPi2, applied in immunoblots from wholekidney membrane preparations, recognized bands at ~85 and~80-85 kDa, respectively, corresponding to published data ( 8, 9 ). Densitometric analysis of blots showed dramatic decreases in the expression of NHE3 and NaPi2 in the hypothyroid groupcompared with controls (12 ± 1.4 vs. 100 ± 7.3 and14.4 ± 4.2 vs. 100 ± 8.4%, respectively; Fig. 1 ).0 d) Y; q% C9 P* R  P0 G
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Fig. 1. Abundance of type 3 Na/H exchanger (NHE3), type 2 Na-Picotransporter (NaPi2), bumetanide-sensitive type 2 Na-K-2Clcotransporter (NKCC2), thiazide-sensitive Na-Cl cotransporter (NCC), -, -, and - subunits of the epithelial Na channel (ENaC),aquaporin-2 (AQP2), and 1 -subunit of Na-K-ATPase bysemiquantitative immunoblotting. Each lane was loaded with an equalamount of total protein extracted from control or methimazole-treatedrat kidneys. ** P P0 M; C3 I1 n  ?3 u0 ~

NKCC2. Antibody to NKCC2 labeled a broad band of ~160-165 kDa in theblots, which agreed with the expected size of 165 kDa ( 8 ). Densitometric analysis showed a marked increase in NKCC2 expression inthe hypothyroid animals (196 ± 18.7 vs. 100 ± 12%; Fig. 1 ). Immunohistochemical staining showed a marked increase in NKCC2 labeling in medullary TAL of hypothyroid kidneys (Fig. 2, A and B ) andunchanged signal intensity in the cortical TAL (not shown).
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Fig. 2. Abundance of NKCC2 ( A, B ) and AQP2( C, D ) by immunohistochemical analysis.Immunoperoxidase signal for NKCC2 is markedly stronger in the apicalpart of the medullary thick ascending limb epithelium (*) inhypothyroid rats ( B ) compared with controls ( A ).Immunoperoxidase signal for AQP2 is markedly stronger in the apicalpart of the collecting duct epithelium in hypothyroid rats (CD in D ) compared with controls (CD in C ) and is moreconcentrated at the luminal cell border; paraffin sections werecounterstained with hematoxylin. Note the smaller size of tubules inthe treated group. Magnification ×1,000.

NCC. Antibody to NCC recognized broad bands at ~160-170 kDa inimmunoblots, as previously reported ( 4 ). Densitometricevaluation revealed no difference in band density (108 ± 1.1 vs.control 100 ± 6%; Fig. 1 ). Immunohistochemical analysis of NCCdistribution did not reveal any changes between groups (Fig. 3, A and B ); signal in both groups was restricted to the apical membrane of DCT cells, confirming previously published data ( 4, 39 ).: c- T2 e8 h: |, C6 ]

Fig. 3. Abundance of NCC ( A, B ) and -ENaC( C - F ) by immunohistochemical analysis oncryostat sections for -ENaC and semithin sections for NCC.Immunofluorescence analysis of NCC shows no obvious differences insubcellular signal distribution or intensity between distal convolutedtubule profiles of a control ( A ) and hypothyroidspecimen ( B ). Immunofluorescence analysis of -ENaC expression shows an equally strong labeling of the apicalmembrane of prinicipal cells in cortical collecting ducts of control( C ) and hypothyroid ( D ) kidneys. There is nodifference in intensity in either profiles of medullary collectingducts ( E control, F hypothyroid); overlay ofimmunostaining (red); and differential interference contrast. A, B : magnification ×1,080; C - F : magnification ×1,000.! V; Z8 B0 w. {) ]3 }' M/ y3 ^' Q3 {

-, -, and -ENaC. Antibodies directed to each of the ENaC subunits recognized bands inthe 80- to 90-kDa range, as previously published ( 18 ). Densitometry of the blots did not show differences between groups ( -ENaC: 111.6 ± 6 vs. control 100 ± 7.4%; -ENaC:102.3 ± 9.7 vs. control 100 ± 12.8%; -ENaC: 103 ± 7.1 vs. control 100 ± 13.3%; Fig. 1 ). Figure 3 showsimmunofluorescence labeling of -ENaC in controls andmethimazole-treated rats (Fig. 3, C - F ). Strong labeling of -ENaC was found in the apical membrane of principal cells in the CCD (Fig. 3, C and D ), whereasstaining in the MCD was distributed more evenly throughout thecytoplasm (Fig. 3, E and F ), which agrees withprevious studies in healthy rats ( 18, 39 ). No differencesin intensity or cellular distribution of -ENaC were detected betweengroups. Anti- - and -ENaC-immunoreactive signals were weaker, butthe distribution corresponded to published data ( 18, 39 ).Differences between groups, however, were not established either(results not shown).& }; Q$ {1 ?0 V6 ?' a
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AQP2. Polyclonal antibody against AQP2 recognized a narrow ~27-kDa band anda broader 35- to 40-kDa band in the blots, which agreed with publisheddata ( 28 ). Densitometric analysis of the blots revealed asharp increase in AQP2 in the hypothyroid group (250.3 ± 27.8 vs.100 ± 15.7%; Fig. 1 ). Immunohistochemistry as well showedenhanced AQP2 signals in CCD and MCD principal cells of the hypothyroidgroup (Fig. 2, C and D ). The association of AQP2 immunostaining with the apical plasma membrane apparently was morepronounced in the hypothyroid kidneys, whereas in controls, the signalshowed a more cytosolic distribution. In both groups, weaker AQP2staining was also recorded in DCT and CNT. However, no changes insignal abundance were observed between groups in these segments.
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1 -Na-K-ATPase. Antibody directed against 1 -Na-K-ATPase recognized anexpected band at ~96 kDa ( 8 ). Densitometric analysis ofthe respective immunoblots showed no significant differences betweenhypothyroid and control animals (98 ± 6.1 vs. 100 ± 2.8%;Fig. 1 ). In the histochemical analysis, no differences were foundbetween groups regarding signal intensity and cellular or zonaldistribution of 1 -Na-K-ATPase (results not shown)./ w1 E- Y2 p6 ]5 L; e
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Calculation of total kidney protein abundance and normalization bycreatinine clearance. As equal amounts of total protein were loaded in each lane forimmunoblots, but less total protein was harvested from kidneys ofmethimazole-treated rats, lanes of treated rats contained greater totalkidney fractions of any given protein. In addition to the rawdensitometric values, we therefore calculated the absolute proteinabundance for each transporter by forming the product of proteincontent of the kidney homogenates and the densitometric intensitylevels. The results showed a dramatic reduction in absolute abundanceof NHE3 and NaPi2, decreased absolute abundance of NCC, -, -, -ENaC, and 1 -Na-K-ATPase, and unchanged absoluteabundance of NKCC2 and AQP2, respectively (Table 2 ). As these values largely reflectchanges in kidney size, it appears essential to relate them to a morefunctional parameter linked to the tubular transport process. Whenprotein abundance was thus normalized by creatinine clearance, NHE3 andNaPi2 were decreased, whereas the remaining transporters were increased(Table 2 ). The increase in abundance was particularly pronounced forNKCC2 and AQP2, which was confirmed on the cellular level byimmunohistochemistry (Fig. 2 ).

Table 2. Calculation of total kidney protein abundance and normalization bycreatinine clearance( K  D2 n7 O; U. ?% G, t

Methimazole-treated, T 3 -substituted rats. In the T 3 -substituted group, serum-free T 3 concentrations were significantly higher than in the group solelyreceiveing methimazole (1.87 ± 0.015 vs. 0.8 ± 0.05 ng/l)but lower than in the euthyroid control group (2.98 ± 0.08 ng/l).T 3 substitution normalized body weight gain (280 ± 5.7 g after 4 wk vs. control 276 ± 4 g), kidney-to-body weight ratio (0.0059 ± 0.0002), creatinine clearance (1.4 ± 0.1 ml · min 1 · g kidneywt 1 ), and FE Na (0.23 ± 0.02%).Densitometric analysis of immunoblots revealed normalization ofabundance of NHE3 (81.5 ± 6.2 vs. control 100 ± 4.1%) andNKCC2 (139.5 ± 4 vs. control 100 ± 3.1%) and largely normalized NaPi2 (56 ± 2 vs. control 100 ± 8%) and AQP2(158.9 ± 6.2 vs. control 100 ± 5.1%).
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DISCUSSION7 Z, M4 i# q: J# u: }6 ~
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In this study, we showed that rats with methimazole-inducedhypothyroidism showed a dramatic deficit in body and kidney weight gain, reduced creatinine clearance, and a marked increase in urinary output and FE Na. This was accompanied by changes in theexpression of proteins that are related to sodium and water transport.Changes were reversible by exogenous T 3 substitution,demonstrating that methimazole itself was without a direct influence ontransporter abundance. Because of the marked differences in kidney sizeand GFR between groups, the conclusion about whether a giventransporter should be considered increased, unchanged, or decreased hasto take into account the alternatives as to how values should be expressed adequately. We thus expressed our values as raw densitometric data (normalized for kidney weight by loading equal amounts of proteinper lane of gel since at first sight it is plausible to assume a linearratio between kidney mass and expression level), or we calculated wholekidney abundances in absolute terms and normalized these to the GFR asestimated by creatinine clearance (since one could argue thattransporter abundances are proportional to the changes in GFR). We alsointerpreted these data relative to immunohistochemical signal intensityas an estimate of the expression of a transporter in any individualepithelial cell.8 R, J9 j3 p; f/ c

All transport proteins investigated in the present study were eitherdecreased or unchanged after methimazole treatment when normalized forkidney mass. Four transporters, however, stood out against the restsince NHE3 and NaPi2 were disproportionately decreased and NKCC2 andAQP2 unchanged. The latter also showed enhanced immunostaining. NCC, -, -, -ENaC, and 1 -Na-K-ATPase were unchangedand did not show differences in immunostaining. Normalized for GFR,NHE3 and NaPi2 showed decreased abundances as well, whereas the otherdistally located transporters were increased, suggesting a change intheir regulation induced by hypothyroidism. In fact, this condition maywell require an enhanced expression of transporters to process a givenfiltrate and may in particular trigger adaptive steps related toimpaired proximal tubular reabsorption. These considerations must beregarded with some reservation, however, as the creatinine clearance asan indicator of GFR is a rather rough marker in rats.

The demonstrated parallel reduction of NHE3 and NaPi2 in hypothyroidkidneys is in agreement with previous data ( 1, 9 ). Decreased NHE3 and NaPi2 expression has furthermore been found in ratswith chronic renal failure ( 29 ), and since we observed increased BUN and serum creatinine levels, this may have influenced thetransporters accordingly. Reduced GFR and luminal flow, which are bothlikely to attenuate luminal Na/H exchange, may have caused a reducedNHE3 expression as well ( 36 ). However, lowered proximal tubular transport has also been observed in hypothyroidism when GFR wasstill normal ( 17 ), which agrees with T 3 actingas a direct stimulator of NHE3 and NaPi2 transcription rate ( 9, 41 ).
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Despite the low GFR, the distal tubular NKCC2 was unchanged inhypothyroidism and increased when normalized for GFR and kidney weightor analyzed by histochemistry, respectively. Impaired proximal tubularreabsorption with a resulting increase in distal sodium delivery may,in fact, be causal for this difference ( 14 ). Parallel stimulation of NKCC2 and AQP2 is probably not related to T 3 deficiency but rather to the enhanced secretion of vasopressin. Thishormone may determine the expression of various sodium transportersincluding NKCC2 in medullary TAL besides regulating water channelabundance in the collecting duct ( 13 ). Effects ofvasopressin on salt reabsorption in medullary TAL have been established(for review, see Ref. 37 ). Effects of hypothyroidism onvasopressin release have been investigated in patients and animalmodels with inconsistent results ( 23, 24, 26, 40 ).However, only plasma vasopressin levels were determined, whichcritically may be subject to short-term variations induced by stressand experimental conditions ( 23 ). To avoid this, wetherefore measured vasopressin excretion since the hormone isrelatively stable in the urine ( 2 ), and we found itsexcretion substantially increased.

Vasopressin may as well raise the expression of other distal tubularproteins such as the ENaC subunits and NCC ( 13, 33 ). Infact, these appeared to be increased when normalized for GFR, but sincethey were not changed in relation to kidney mass and histochemicalsignal intensity, we consider their definitive stimulation in thehypothyroid organism unlikely. Alternatively, a modified vasopressinresponsiveness may be considered in hypothyroidism since a diminishedV 2 receptor-mediated release of cAMP has been reported inkidneys under this condition ( 19 ), and related findings were reported in hepatocytes ( 30 ). The efficiency ofvasopressin may as well be reduced by a diminished concentration ofmedullary solutes in hypothyroidism, hereby aggravating urinaryconcentration deficit and polyuria ( 38 ).0 u! Q2 Z/ b( H& S2 b
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Another potential cause for hypothyroid sodium losses may be a reducedactivity of Na-K-ATPase and the resulting decline of ion gradientdriving forces as reported in proximal tubules and collecting ducts( 3 ). Decreased Na-K-ATPase activity has been attributed toa selective decrement of -Na-K-ATPase abundance ( 22 ).In accordance with this, we found the abundance of the 1 -subunit of Na-K-ATPase only lowered to the same extentas kidney size and unchanged in immunohistochemical staining. Inaddition to its direct stimulatory effect, T 3 also enhancesNa-K-ATPase activity by increasing the sensitivity of target cells tomineralocorticoids ( 3 ). Besides Na-K-ATPase, NCC and ENaCare also aldosterone-dependent products, and a permissive role ofT 3 on the action of mineralocorticoids must thus beconsidered. Because the products were not altered in our hands,however, one may only speculate that mineralocorticoid-dependent adaptation under additional challenges may be impaired by this deficitas observed elsewhere ( 35 ).+ a% @7 c" P9 ^. A* J5 z4 L/ I! l# P

Finally, structural changes of the hypothyroid kidney may contribute tothe presumed deficit in late distal sodium reabsorption. We couldconfirm data showing that renal growth is proportionately more affectedthan total body growth ( 5, 6 ). Measuring the length ofmicrodissected tubules from hypothyroid rat kidneys had previouslyrevealed that renal growth reduction mainly resulted from a decrementin length of the proximal and distal renal tubule, whereas glomerulargrowth was retarded only to the extent of total body growth( 6 ). Assuming a distal filtrate delivery as high inhypothyroid rats as in controls ( 32 ), it therefore seems likely that distal sodium handling may be related to disproportionately shortened distal convolutions.# B$ |# h3 V- ^1 ?" ?

Despite the kidney's need for thyroid hormone to maintain sodiumbalance ( 42 ), we found no indication for a disturbedbalance in this study consistent with previous studies (reviewed inRef. 25 ). Additonal manipulations such as asodium-deficient diet, saline or water loading, or others, however,cause excessive natriuresis in the hypothyroid organism ( 7, 16, 21, 42 ).

In summary, we demonstrate that hypothyroidism in rats is associatedwith an altered abundance of renal sodium entry pathways along with adecrement in creatinine clearance, increased FE Na, increased vasopressin excretion, and polyuria. A proximal decrease ofNHE3 and NaPi2 is likely to be related to the low T 3 levels as well as the reduced GFR. Distal straight tubule and collecting ductincreases in NKCC2 and AQP2 probably reflect a vasopressin-induced adaptation of the tubule to preserve water and sodium. The observed changes were largely corrected by substitution of exogenousT 3., s  w8 n( V- Q

ACKNOWLEDGEMENTS! }1 {6 f  }- E" Z! W

We express our gratitude to P. Exner and S. Diederich for thegenerous help measuring urinary vasopressin and J. Biber for providingthe NaPi2 antibody.
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