Segment-specific ENaC downregulation in kidney of rats with lithium-induced NDI - 《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 13:48 |只看该作者 |倒序浏览 |打印

作者：Jakob Nielsen, Tae-Hwan Kwon, Jeppe Praetorius, Young-Hee Kim, Jørgen Frøkiær, Mark A. Knepper,  Søren Nielsen作者单位：1 The Water and Salt Research Center, University of Aarhus, DK-8000 Aarhus C, Denmark; Department of Physiology, School of Medicine, Dongguk University, Kyungju 780-71 Korea; and Laboratory of Kidney and Electrolyte Metabolism, National Heart Lung and Blood Institute, National Institutes of Health,
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      【摘要】! u2 `7 T6 {  e* W# G
   Lithium-induced nephrogenic diabetes insipidus is associated with increased renal sodium excretion in addition to severe urinary concentrating defects. However, the molecular basis for this altered renal sodium excretion remains undefined. The amiloride-sensitive sodium channel (ENaC) is expressed in the renal connecting tubule and collecting duct and is essential in renal regulation of body sodium balance and blood pressure. We hypothesized that dysregulation of ENaC subunits may be responsible for the increased sodium excretion associated with lithium treatment. Lithium treatment for 28 days resulted in severe polyuria, increased fractional excretion of sodium, and increased plasma aldosterone concentration. Immunoblotting revealed that lithium treatment induced a marked decrease in the protein abundance of -ENaC and -ENaC in the cortex and outer medulla. Moreover, immunohistochemistry and laser confocal microscopy demonstrated an almost complete absence of -ENaC and -ENaC labeling in cortical and outer medullary collecting duct, which was not affected by dietary sodium intake. In contrast, immunohistochemistry showed increased apical labeling of all ENaC subunits in the connecting tubule and inner medullary collecting duct in rats on a fixed sodium intake but not in rats with free access to sodium. Except for a modest downregulation of the thiazide-sensitive Na-Cl cotransporter, the key renal sodium transporters upstream from the connecting tubule (including the 1 -subunit of Na-K-ATPase, type 3 Na/H exchanger, and Na-K-2Cl cotransporter) were unchanged. These results identify a marked and highly segment-specific downregulation of -ENaC and -ENaC in the cortical and outer medullary collecting duct, chief sites for collecting duct sodium reabsorption, in rats with a lithium-induced increase in fractional excretion of sodium.
      【关键词】 aldosterone collecting duct epithelial sodium channel hypertension nephrogenic diabetes insipidus sodium reabsorption sodium wasting
   LITHIUM IS AN IMPORTANT DRUG widely used in the management of bipolar affective mood disorders ( 50 ). Unfortunately, lithium treatment is associated with decreased urinary concentrating ability in up to 50% of patients receiving lithium therapy and overt polyuria in 20% of patients ( 3, 8 ). The impaired urinary concentration ability and polyuria have been associated with impaired V 2 receptor-mediated vasopressin responsiveness ( 12 ) and decreased protein abundances of aquaporin-2 (AQP2) and aquaporin-3 (AQP3) in collecting duct principal cells ( 26, 30 ).

Lithium treatment is also associated with a concomitant increase in urinary sodium excretion ( 5, 39, 47 ). However, the mechanism for sodium wasting is still incompletely understood and the molecular basis remains undefined. Studies have implicated the aldosterone-responsive distal renal tubule and collecting duct segments in the increased urinary sodium excretion in response to lithium treatment. Specifically, physiological studies have shown that chronic lithium treatment induces decreased responsiveness of sodium reabsorption to mineralocorticoids and amiloride ( 5, 46, 48 ). Because the amiloride-sensitive sodium channel (ENaC) is a known target for aldosterone-stimulated sodium reabsorption in the renal collecting duct ( 31 ), these studies raise the possibility that ENaC is also the target for the natriuretic effects of lithium. Compatible with distal effects of lithium, Kwon et al. ( 26 ) demonstrated that key apical sodium transporters expressed in the renal tubule segments proximal to the connecting tubule were not downregulated, including transporters in the proximal tubule [type 3 Na/H exchanger (NHE3) and the 1 -subunit of Na-K-ATPase] and thick ascending limb (Na-K-2Cl cotransporter; NKCC2) despite an increased urinary sodium excretion. We therefore hypothesize that altered expression and regulation of ENaC subunits are importantly involved in the increased urinary sodium excretion in rats chronically treated with lithium.1 g5 i& {& }" O: V1 [
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ENaC is expressed in connecting tubule cells and principal cells of the collecting duct ( 19 ). It is the principal transporter of sodium across the apical plasma membrane and reabsorbs a large fraction of the sodium delivered from the distal convoluted tubule in the connecting tubule and cortical collecting duct ( 2, 43 ). ENaC is important in the regulation of sodium balance, extracellular fluid volume, and blood pressure ( 17 ). ENaC is a heteromeric protein with three homologous subunits, i.e., -, -, and -ENaC ( 9 ), and is characterized by a cation selectivity for sodium and lithium ( 37 ). Expression of ENaC subunits in Xenopus laevis oocytes and the generation of gene knockouts of the individual subunits in mice have demonstrated that altered expression of any of the three subunits has significant effects on the multimeric ENaC protein sodium transport capacity ( 6, 9, 22, 34 ). Accordingly, regulation of sodium reaborption by ENaC mediated by hormones such as vasopressin and aldosterone ( 17 ) is associated with characteristic alterations in the expression of the individual ENaC subunits ( 15, 31 ). Chronic vasopressin infusion in naturally vasopressin-deficient Brattleboro rats resulted in significantly increased abundances of all three ENaC subunits, whereas 7-day water restriction in Sprague-Dawley rats results in significantly increased abundances of only -ENaC and -ENaC ( 15 ). Chronic aldosterone infusion to Sprague-Dawley rats increases the protein abundance of -ENaC. Moreover, aldosterone causes a mobility shift of -ENaC from an 85- to a 70-kDa band without a change in total -ENaC protein abundance ( 31 ). The appearance of the 70-kDa form of -ENaC in response to aldosterone is putatively due to a channel-activating proteolytic cleavage ( 51 ). The same changes are observed in chronically sodium-restricted rats in addition to a significant downregulation of the -ENaC subunit ( 32 ). ENaC is also subjected to regulation by trafficking. In normal rat kidney, we have demonstrated that immunolabeling of -ENaC is mainly present at the apical domains of the principal cells, whereas the labeling of -ENaC and -ENaC is associated with intracellular vesicles dispersed in the entire cytoplasm with sparse labeling of the apical plasma membrane ( 19 ). Elevated plasma aldosterone concentration is associated with a markedly increased apical expression of -ENaC and -ENaC, with a lesser effect on -ENaC already present in the apical domain ( 28, 31 ). This suggests that there are also differences in the regulation of the subcellular localization of ENaC subunits. In addition to the described regulatory mechanisms, ENaC is also subjected to regulation by the tubular sodium load, intraluminal flow rate, intracellular pH, and angiotensin II ( 7, 14, 36, 38, 40 ).

The purpose of this study was to directly investigate the effect of chronic lithium treatment on the regulation of ENaC subunit protein abundance and the segmental and subcellular localization to elucidate the underlying molecular mechanisms responsible for the increased urinary sodium excretion associated with prolonged lithium treatment.9 z9 Q& O9 ?( k. k) T' ^5 |
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METHODS& \; u* A1 ^, J2 i8 }5 D

Animal protocol 1: low-dose lithium treatment with a fixed sodium intake. Male Wistar rats ( n = 12, body wt 190-210 g, Møllegaard Breeding Center, Skensved, Denmark) received daily food rations of a food mix consisting of 20 g of ground rat chow (Altromin 1320, Petersen, Ringsted, Denmark), 20 ml tap water, and an additional 0.25 mmol NaCl (total content of sodium 2.5 mmol Na/day). The lithium-treated rats received 0.8 mmol LiCl (L 4408, Sigma) added to the food mixture. In addition to the water in the food mixture, the rats had free access to a water bottle. For the first 16 days, the rats were housed individually in normal rat cages, and each received 40 g of the food mix/day. During the last 12 days, the rats were housed in metabolic cages and pair fed. The controls were offered only the amount (of the 40 g offered) that the lithium-treated rats ate, which averaged 33 g of the food mix. The daily water intake and food intake were monitored throughout the study. Urine was analyzed for concentrations of sodium, potassium, creatinine, and urea and for the measurement of urinary osmolality. During the entire experiment, there was a 12:12-h artificial light-dark cycle, a temperature of 21 ± 2°C, and humidity of 55 ± 2%. The food was administered daily 1 h before the dark cycle began. On day 28, the rats were anesthetized with halothane (Halocarbon Laboratories, River Edge, NJ), and a large laparotomy was made. Blood was collected from the inferior vena cava. The blood was prepared to be analyzed for sodium, potassium, creatinine, urea, osmolality, and plasma aldosterone concentration. The right kidney was rapidly removed, dissected into regions (cortex, inner stripe of outer medulla and inner medulla), and processed for immunoblotting as described below. The left kidney was perfusion fixed as described below." c  F: L8 S, I( g0 p; s7 j
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Animal protocol 2: high-dose lithium treatment with free access to sodium. Male Wistar rats were used ( n = 12, Møllegaard Breeding Center). Lithium chloride was added to rat chow to give a concentration of 40 or 60 mmol lithium/kg dry food (Altromin 1320, Petersen) as previously described ( 12, 30 ). Rats received food containing 40 mmol lithium/kg dry food for the first 7 days and thereafter 60 mmol lithium/kg dry food. All rats had access to a NaCl licking-block to replace sodium loss and avoid lithium intoxication. This protocol has been reported to give a plasma lithium concentration of 0.70 ± 0.09 mM ( 12 ) and a similar protocol with 80 mmol lithium/kg dry food and free access to sodium resulted in a plasma lithium concentration of 0.89 ± 0.13 mM ( 49 ). The rats had free access to a water bottle. The rats were housed individually in normal rat cages for the first 20 days and then in metabolic cages for the last 7 days of the study to allow measurements of urine production. On the last day of the study, the rats were killed and kidneys were perfusion fixed as described below. The studies complied with the Danish regulations (Danish Ministry of Justice) for the care and use of experimental animals.

Semiquantitative immunoblotting. The procedure was similar to what has been described in detail previously ( 24, 45 ) and is summarized briefly in the following. The dissected renal cortex, inner stripe of the outer medulla, and inner medulla were homogenized (Ultra-Turrax T8 homogenizer, IKA Labortechnik, Staufen, Germany) in ice-cold isolation solution containing 0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, 8.5 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride, pH 7.2. To remove large cellular debris and nuclei, the homogenates were centrifuged at 4,000 g for 15 min at 4°C, and the supernatant was pipetted off and kept on ice for further processing. The total protein concentration was measured (Pierce BCA protein assay reagent kit, Pierce, Rockford, IL), and all samples were adjusted with an isolation solution to reach the same final protein concentrations, solubilized at 65°C for 15 min in Laemmli sample buffer, and then stored at -20°C. To confirm equal loading of protein, an initial gel was stained with Coomassie blue as described previously ( 45 ). SDS-PAGE was performed on 9 or 12% polyacrylamide gels. The proteins were transferred from the gel electrophoretically (Bio-Rad Mini Protean II) to nitrocellulose membranes (Hybond ECL RPN3032D, Amersham Pharmacia Biotech, Little Chalfont, UK). After transfer, the blots were blocked with 5% milk in PBS-T (80 mM Na 2 HPO 4, 20 mM NaH 2 PO 4, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated overnight at 4°C with primary antibodies. The sites of antibody-antigen reaction were visualized with horseradish peroxidase-conjugated secondary antibodies (P447 or P448, diluted 1:3.000; DAKO, Glostrup, Denmark) with an enhanced chemiluminescence (ECL or ECL ) system and exposed to photographic film (Hyperfilm ECL, RPN3103K, Amersham Pharmacia Biotech). The band densities were quantitated by scanning the films and normalizing the densitometry values to facilitate comparisons. Results are listed as the relative, and not absolute, band densities between the groups, hence the term semiquantitative immunoblotting.  I9 w) i& \1 s$ b( `: {: v

Immunohistochemistry. A perfusion needle was inserted into the abdominal aorta, and the inferior vena cava was cut to establish an outlet after blood sampling. Blood was flushed from the kidneys with cold 0.01 M PBS (pH 7.4) for 15 s, before a switch to cold 3% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 3 min. The kidney was removed, and the midregion was sectioned into 2- to 3-mm transverse sections and immersion fixed additionally for 1 hour, followed by 3 x 10-min washes with 0.1 M cacodylate buffer, pH 7.4. The tissue was dehydrated in graded ethanol and left overnight in xylene. After the tissue was embedded in paraffin, 2-µm sections were cut on a rotary microtome (Leica Microsystems, Herlev, Denmark).
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For immunolabeling, the sections were dewaxed with xylene and rehydrated with graded ethanol. Sections had endogenous peroxidase activity blocked with 0.5% H 2 O 2 in absolute methanol for 10 min. Using a microwave oven, the sections were boiled in a target-retrieval solution (1 mM Tris, pH 9.0, with 0.5 mM EGTA) for 10 min. After cooling, non-specific binding was blocked with 50 mM NH 4 Cl in PBS for 30 min followed by 3 x 10 min with PBS blocking buffer containing 1% BSA, 0.05% saponin, and 0.2% gelatin. The sections were incubated with primary antibody (diluted in PBS with 0.1% BSA and 0.3% Triton X-100) overnight at 4°C. The sections were washed 3 x 10 min with PBS wash buffer containing 0.1% BSA, 0.05% saponin, and 0.2% gelatin and incubated with horseradish peroxidase-conjugated secondary antibody (DAKO P448, goat anti-rabbit IgG) for 1 h at room temperature. After 3 x 10-min rinses with PBS wash buffer, the sites of antibody-antigen reaction were visualized with a brown chromogen produced within 10 min by incubation with 0.05% 3,3'-diaminobenzidine tetrachloride (Kemen Tek, Copenhagen, Denmark) dissolved in distilled water with 0.1% H 2 O 2. Mayer's hematoxylin was used for counterstaining, and after dehydration coverslips were mounted with hydrophobic medium (Eukitt, Kindler, Freiburg, Germany). For sections prepared for immunofluorescence, a secondary fluorescent antibody was used (goat anti-rabbit IgG, Alexa Fluor 488, 11008; and goat anti-mouse IgG, Alexa Fluor 546, 11003, Molecular Probes, Eugene, OR). After a 1-h incubation at room temperature, coverslips were mounted with hydrophilic mounting media containing an antifading reagent ( n -propyl-gallat, P-3101, Sigma, St. Louis, MO). Light microscopy was carried out with a Leica DMRE (Leica Microsystems). Laser confocal microscopy was carried out with a Leica TCS-SP2 laser confocal microscope (Leica, Heidelberg, Germany).
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Antibodies. Rabbit polyclonal antibodies to the following renal sodium transporters were utilized: NHE3 of the proximal tubule ( 16 ); NKCC2 of the thick ascending limb ( 24 ); the thiazide-sensitive Na-Cl cotransporter (NCC) of the distal convoluted tubule ( 25 ); and the ENaC subunits -ENaC, -ENaC, and -ENaC in the connecting tubule and collecting duct ( 31 ). The antisera were affinity purified against the immunizing peptides as previously described ( 24, 25 ). Specificity of the antibodies has been demonstrated by showing unique peptide-ablatable bands on immunoblots and a specific labeling by immunocytochemistry. Additional antiserum against -ENaC used for immunocytochemistry was kindly provided by Dr. A. K. Vinnikova (McGuire Veterans Affairs Medical Center, Richmond, VA), and the monoclonal 1 -subunit of Na-K-ATPase was generously provided and characterized by Dr. D. M. Fambrough (Johns Hopkins Univ. Medical School) ( 23 ).$ ~% ^! |( c* b
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Presentation of data and statistical analyses. Quantitative data are presented as means ± SE. Statistical comparisons were accomplished by unpaired t -test (when variances were the same) or by Mann-Whitney rank-sum test (when variances were significantly different between groups). P values considered statistically significant.: a) i4 {( d, B* ^+ E
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Lithium-treated rats had increased fractional excretion of sodium. Consistent with previous observations ( 26, 30 ), the lithium-treated rats (see METHODS ) developed polyuria (156 ± 24 vs. 24 ± 1 ml/day, P ± 22 vs. 895 ± 42 mosmol/kgH 2 O, P Table 1 ). The lithium-treated and control rats consumed equal amounts of food (i.e., the same sodium and potassium intake) and, as expected, urinary sodium and potassium excretion rates were not different ( Table 1 ). However, the lithium-treated rats had a marked increase in the fractional excretion of sodium (0.80 ± 0.02 vs. 0.60 ± 0.03%, P the lithium-treated rats had decreased creatinine clearance (1.49 ± 0.12 vs. 1.86 ± 0.08 ml/min, P and a decreased filtered load of sodium (207 ± 18 vs. 262 ± 11 µmol/min, P that lithium treatment is associated with an impairment of the tubular reabsorption of filtered sodium, thereby producing natriuresis and extracellular fluid volume contraction despite markedly increased plasma aldosterone concentrations (11.5 ± 2.2 vs. 0.35 ± 0.06 nM, P

Table 1. Physiological data of effect of lithium treatment

Lithium-treated rats had significantly reduced expression of -ENaC and -ENaC in cortical and outer medullary collecting duct principal cells. To investigate the effect of lithium treatment on ENaC subunit protein abundances in the cortex, inner stripe of the outer medulla, and inner medulla, semiquantitative immunoblotting was carried out. Using protein from cortical homogenates, immunoblots revealed markedly decreased band densities of -ENaC and -ENaC (both 85- and 70-kDa bands of -ENaC) in the lithium-treated rats compared with control rats ( Fig. 1 ). On the other hand, the band density of -ENaC was not significantly changed. The analyses of normalized band densities are shown in Table 2. The changes in the inner stripe of the outer medulla were similar to the changes in the cortex, namely, markedly downregulated -ENaC and -ENaC (only the 85-kDa band was observed on the immunoblot shown, and a negligible 70-kDa band was observed with longer film exposure) and no change in -ENaC protein expression despite the marked elevation of plasma aldosterone concentration ( Fig. 2 ).5 e1 }: ^2 L  @
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Fig. 1. Semiquantitative immunoblots using protein prepared from cortical homogenates from control and lithium-treated rats. Equality of protein amount loaded was ensured by Coomassie blue staining of gel after electrophoresis. The -subunit of the epithelial sodium channel ( -ENaC) band is seen at 85 kDa and is unchanged. The band is a composite of several thin bands believed to represent different glycosylated forms of -ENaC. The -ENaC band at 85 kDa was significantly downregulated. This band is also presented as several thin bands representing different glycosylated forms of -ENaC. -ENaC is seen as a narrow band around 85 kDa and a broader band around 70 kDa. Both the 85- and 70-kDa bands are significantly downregulated. * P# c$ Y, Z8 M; n0 K# z( E

Table 2. Densitometric analysis of immunoblots from lithium-treated rats
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Fig. 2. Semiquantitative immunoblots using protein prepared from inner stripe of outer medulla homogenates from control and lithium-treated rats. There is no change in the -ENaC band at 85 kDa. Both the -ENaC and 85-kDa band of -ENaC are significantly downregulated in lithium-treated rats. The 70-kDa band density is negligible and only detected weakly on a longer exposure film. * P  p$ X: H  R  F8 [" N4 w

In addition to the changes in protein abundances of the ENaC subunits, the sodium channel is also regulated by intracellular trafficking. Increased plasma aldosterone concentration mediates a change in the subcellular localization of ENaC from a dispersed cytoplasmic distribution to the apical plasma membrane domains ( 28, 31 ). To investigate whether lithium treatment affected this response, we carried out immunoperoxidase labeling and immunofluorescence labeling of tissue sections for microscopic analysis. Striking findings were observed for -ENaC and -ENaC. In the lithium-treated rats, the cortical collecting ducts showed markedly reduced labeling of -ENaC and -ENaC with no apparent apical labeling ( Fig. 3, B and D, respectively). In control rats, there was dispersed cytoplasmic labeling of both -ENaC and -ENaC in the collecting duct principal cells ( Fig. 3, A and C, respectively). Similarly, in the outer medullary collecting duct (both inner and outer stripe), labeling of both -ENaC (not shown) and -ENaC was decreased in lithium-treated rats and dispersed in control rats ( Fig. 3, E and F ). To confirm that the tubule segments were cortical collecting duct and not connecting tubule, double labeling with -ENaC and calbindin-D 28k was carried out and analyzed by laser scanning confocal microscopy. As described below, calbindin-D 28k is expressed in connecting tubule cells. The tubule segments showing dispersed -ENaC labeling (green) in control rats ( Fig. 3 G ) and no labeling in lithium-treated rats ( Fig. 3 H ) were all negative for calbindin-D 28k (red), thus confirming the tubule to be the cortical collecting duct. In contrast, there were no major changes in the subcellular localization of -ENaC immunolabeling in the cortical and outer medullary collecting duct in lithium-treated rats compared with control rats (not shown). The labeling of -ENaC remained dispersed in the cytoplasm, with some apical labeling in the cortex but not in the outer medulla of both control rats and lithium-treated rats.
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Fig. 3. Immunoperoxidase and immunofluorescence microscopy of - and -ENaC in collecting duct. Labeling of - and -ENaC is strong and dispersed in the cytoplasm of principal cells of the cortical collecting duct (CCD; A and C ) in control rats. However, the labeling intensity is strikingly reduced in the cytoplasm of principal cells in the same segments of lithium-treated rats ( B and D ). In the outer medullary collecting duct (OMCD), similar changes were observed ( E and F ). With double labeling, the tubule segment with dispersed -ENaC labeling (green) in control rats ( G ) does not show any calbindin-D 28k labeling (red), confirming that the tubule is CCD. In the lithium-treated rats, the tubule segment is unlabeled with both -ENaC and calbindin-D 28k ( H ), thus confirming that the tubule is CCD. (See Fig. 4 for examples of positive calbindin-D 28k labeling.) The tubular structures are visualized by differential interference contrast (DIC). Arrows indicate dispersed intracellular labeling. Scale bars, 10 µm.* L( }! u0 l% _/ Y# {' t( `* _
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Fig. 4. Immunoperoxidase and immunofluorescence microscopy of - and -ENaC in connecting tubule (CNT). In the CNT, - and -ENaC labeling is dispersed in the cytoplasm of CNT cells in the control rats ( A and C ), whereas lithium-treated rats show a distinct apical labeling ( B and D ). Calbindin-D 28k was used as a marker for the CNT, where it is abundantly expressed. The colocalization of -ENaC and calbindin-D 28k in the same cells is observed for both control ( E ) and lithium-treated rats ( F ). The -ENaC labeling (green) is dispersed in CNT cells of control rats ( E and H ) and distinctly apical in the lithium-treated rats ( F and J ). Calbindin-D 28k labeling (red) is dispersed in the cells labeling with -ENaC ( G and I ). Arrows indicate dispersed intracellular labeling. Arrowheads indicate apical labeling. Scale bars, 10 µm.

Lithium-treated rats had increased apical ENaC subunit expression in connecting tubule cells. In contrast to the significant downregulation of -ENaC and -ENaC in the cortical and outer medullary collecting duct, immunocytochemistry revealed a distinct apical labeling of -ENaC and -ENaC ( Fig. 4, B and D, respectively) in the connecting tubule cells in lithium-treated rats. Labeling of -ENaC and -ENaC in control rats was dispersed throughout the cytoplasm of connecting tubule cells ( Fig. 4, A and C, respectively). The labeling of -ENaC was unchanged and showed both apical and dispersed labeling in control and lithium-treated rats (not shown). To confirm that the distinct apical labeling observed was located in connecting tubule cells, double labeling with -ENaC and calbindin D 28k was carried out and analyzed by laser scanning confocal microscopy. Calbindin-D 28k was used as a marker for connecting tubule, where it is abundantly expressed, although it is also expressed at much lower levels in late distal convoluted tubule and initial collecting duct in rat kidney ( 27, 44 ). Figure 4 shows the colocalization of -ENaC (green) and calbindin-D 28k (red) in the same cells ( Fig. 4, E and F ). In the lithium-treated rats, most of the cells displaying distinct apical -ENaC labeling in cortex were strongly colabeled with calbindin-D 28K, demonstrating that these cells are connecting tubule cells ( Fig. 4, I and J ). In control rats, the labeling of -ENaC was dispersed in cells coexpressing high levels of calbindin-D 28k ( Fig. 4, G and H ).

Lithium-treated rats had increased expression of -ENaC and -ENaC in inner medullary collecting duct cells, but unchanged -ENaC expression. Immunoblotting of protein from kidney inner medulla revealed a significantly increased protein abundance of -ENaC and the 70-kDa form of -ENaC in lithiumtreated rats compared with controls ( Fig. 5 ). These two changes are compatible with changes seen in response to increased plasma aldosterone concentrations ( 31 ). In contrast, there were no changes in band densities of -ENaC and the 85-kDa band of -ENaC, although the total abundance of -ENaC was increased due to the appearance of the 70-kDa form of -ENaC (the sum of the normalized band densities of 85- and 70-kDa: 1.00 ± 0.04 vs. 1.88 ± 0.07, P The increased expression of -ENaC is compatible with changes seen in response to increased plasma vasopressin concentrations. Immunohistochemistry showed distinct apical labeling of all three ENaC subunits in inner medullary collecting duct cells. Consistent with immunoblotting, the immunolabeling intensity of -ENaC in the inner medulla was markedly increased and showed distinct apical labeling ( Fig. 6 B ). The apical labeling of -ENaC (not shown) and -ENaC ( Fig. 6 D ) in the lithium-treated rats was predominant only in the middle region of the inner medulla. In contrast, labeling in control rats was only weak and dispersed in inner medullary collecting duct cells ( Fig. 6, A and C ).
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Fig. 5. Semiquantitative immunoblots using protein prepared from inner medullary homogenates prepared from control and lithium-treated rats. There is a significant increase in -ENaC protein abundance, whereas -ENaC and the 85-kDa band of -ENaC are unchanged. However, the 70-kDa band of -ENaC is significantly increased in the lithium-treated rats. * P3 g7 h! B* F; ^8 }8 T$ I
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Fig. 6. Immunoperoxidase microscopy of -ENaC and -ENaC in inner medulla. The labeling of -ENaC is weak and dispersed in the cytoplasm of inner medullary collecting duct cells in control rats ( A ), whereas the lithium-treated rats show distinct apical labeling ( B ) in addition to dispersed cytoplasmic labeling. The labeling of -ENaC is weak and dispersed in the cytoplasm in control rats ( C ) but distinctly apical in the lithium-treated rats ( D ). The apical labeling of ENaC subunits was predominant only in the middle region of the inner medulla. Arrows indicate dispersed intracellular labeling. Arrowheads indicate apical labeling. Scale bars, 10 µm.- F6 ^& |$ Q* s/ f5 n& M% s' x8 E

Lithium-treated rats with free access to sodium also showed markedly downregulated -ENaC and -ENaC in cortical and outer medullary collecting duct. In previous studies with chronic lithium treatment, the rats had free access to NaCl to compensate for increased renal sodium excretion caused by lithium ( 26, 48 ). To confirm that the downregulation of -ENaC and -ENaC in cortical and outer medullary collecting duct is not related to secondary changes caused by a fixed sodium intake, we analyzed tissue sections labeled with -ENaC and -ENaC from lithium-treated rats with free access to sodium (see METHODS ). These rats also showed markedly reduced labeling intensity of -ENaC (not shown) and -ENaC in cortical ( Fig. 7 B ) and outer medullary collecting duct ( Fig. 7 D ) compared with control rats ( Fig. 7, A and C, respectively). These findings are identical to the labeling patterns seen in lithium-treated rats with a fixed sodium intake ( Fig. 3 ) and support the view that the downregulation of -ENaC and -ENaC in these segments is a primary effect of lithium treatment. Interestingly, there was much less distinct apical labeling of ENaC in the connecting tubule and inner medullary collecting duct in the lithium-treated rats with free access to sodium.
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Fig. 7. Immunoperoxidase microscopy of -ENaC in CCD and OMCD of lithium-treated rats with free access to sodium. Labeling of -ENaC is dispersed in the cytoplasm of CCD ( A ) and OMCD ( C ) principal cells in control rats. Lithium-treated rats show a marked reduction in the labeling intensity in the same segments ( B and D, respectively). Arrows indicate dispersed intracellular labeling. Scale bar, 10 µm.: [) @& P/ F. B9 l0 g: W
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Lithium clearance and fractional excretion of lithium in lithium-treated rats. To evaluate how the differences in ENaC regulation affected the renal handling of lithium, we measured plasma and urinary parameters to calculate the renal clearance of creatinine and lithium and the fractional excretion of lithium in two different sets of rats. Rats treated with a low dose of lithium and with a fixed sodium intake (less sodium intake; protocol 1 ) had plasma lithium concentrations of 0.61 ± 0.02 mM. Rats treated with a high dose of lithium and with free access to sodium (more sodium intake; protocol 2 ) had a similar plasma lithium concentration of 0.61 ± 0.09 mM. The creatinine clearance in lithium-treated rats with a fixed sodium intake was 1.32 ± 0.19 ml/min, and lithium clearance was 0.59 ± 0.03 ml/min. The ratio of lithium clearance to creatinine clearance as a measure of the fractional excretion of lithium was 0.51 ± 0.09. In contrast, rats with free access to sodium had a creatinine clearance of 1.92 ± 0.11 ml/min, lithium clearance of 1.57 ± 0.29 ml/min, and fractional excretion of lithium of 0.82 ± 0.14. Therefore, the decreased clearance and fractional excretion of lithium in rats with a fixed sodium intake (less sodium intake; protocol 1 ) may contribute to the similar plasma lithium concentrations between the two groups, despite the fact that their lithium intake was lower. The presence of similar plasma lithium concentrations suggests that the differences in ENaC labeling observed in the connecting tubule ( Fig. 4 ) and inner medullary collecting duct ( Fig. 6 ) of lithium-treated rats with a fixed sodium intake and free sodium intake are largely dependent on the differences in sodium intake.
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Lithium-treated rats showed decreased NCC expression, whereas expression of NHE3, NKCC2, and 1 -subunit of Na-K-ATPase was unaffected or increased. To confirm that the increased fractional excretion of sodium in lithium-treated rats was not associated with decreased protein expression of other major cortical sodium transporters located proximal to ENaC expression sites (i.e., connecting tubule and collecting duct), additional immunoblots were carried out using antibodies against NHE3, NKCC2, NCC, and the 1 -subunit of the Na-K-ATPase ( Fig. 8; summary of normalized band densities in Table 2 ). As expected, these transporters were not significantly changed except for NCC in the cortex, which was significantly downregulated in lithium-treated rats. In the inner stripe of the outer medulla, there was no change in NKCC2 or Na-K-ATPase 1 -subunit protein expression. In the inner medulla, the Na-K-ATPase 1 -subunit was significantly upregulated. Immunolabeling of tissue sections with the 1 -subunit of the Na-K-ATPase showed upregulation in the same region where ENaC was expressed in the apical cell domain of inner medullary collecting duct cells (not shown). Consistent with previous studies, the AQP2 water channel was also significantly downregulated in both the cortex and inner medulla of lithium-treated rats, whereas AQP1 was unchanged in the cortex (summary of normalized band densities in Table 2; immunoblots not shown).) F. _2 z% f3 y. g* S$ A. R

Fig. 8. Semiquantitative immunoblots using protein prepared from homogenates from the cortex ( A ), inner stripe of outer medulla ( B ), and inner medulla ( C ). In the cortex, there is no change in the protein abundance of type 3 Na/H exchanger (NHE3), Na-K-2Cl cotransporter (NKCC2), and Na-K-ATPase 1 -subunit, but that for the Na-Cl cotransporter (NCC) shows a significant reduction in the lithium-treated rats. In the inner stripe of the outer medulla, there is no change in NKCC2 and Na-K-ATPase 1 -subunit. In the inner medulla, there is a significant increase in the protein abundance of the Na-K-ATPase 1 -subunit. * P" N# Z1 {- }- d! v
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DISCUSSION
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The results of this study demonstrate a highly segment-specific downregulation of -ENaC and -ENaC in cortical and outer medullary collecting duct in lithium-treated rats with reduced tubular reabsorption of filtered sodium. These changes, affecting chief sites of collecting duct sodium reabsorption, are consistent with previous studies showing reduced amiloride-sensitive sodium reabsorption in the collecting duct in rats treated with lithium ( 46 ). Moreover, NCC expression in the distal convoluted tubule was also reduced, which may participate in the reduction in distal tubular sodium reabsorption. Increased apical ENaC expression was noted in connecting tubule and inner medullary collecting duct in rats with fixed sodium intake but not with free access to sodium and is therefore likely to be a compensatory mechanism limiting renal sodium loss. There was no reduction in the expression of other major renal sodium transporters. Thus the marked downregulation of -ENaC and -ENaC is likely to represent the key molecular basis for the frequently observed sodium wasting associated with lithium treatment of common bipolar affective disorders.

Significant downregulation of -ENaC and -ENaC in the cortical collecting duct and outer medullary collecting duct. Lithium-induced nephrogenic diabetes insipidus (NDI) is associated with polyuria, decreased urine concentration, and increased urinary sodium excretion, despite high plasma vasopressin concentration ( 18 ) and plasma aldosterone concentration ( 20 ). A significant downregulation of vasopressin-regulated water channels AQP2 and AQP3 ( 26, 30 ) appears to play a crucial role in the development of lithium-induced polyuria. In contrast, the abundance of several major renal sodium transporters (NHE3, NKCC2, and the 1 -subunit of Na-K-ATPase) expressed in the nephron proximal to the connecting tubule was increased or unchanged, despite an increase in urinary sodium excretion ( 26 ). This led us to examine the changes in protein abundance and subcellular localization of the ENaC subunits expressed in the connecting tubule and collecting duct to explore the molecular mechanisms of natriuresis associated with lithium-induced NDI. As described above, we identified severe downregulation of -ENaC and -ENaC in the cortical collecting duct and outer medullary collecting duct in lithium-treated rats displaying reduced fractional reabsorption of sodium. As discussed below, downregulation of -ENaC and -ENaC without changes in -ENaC is likely to play a key role in the sodium wasting associated with lithium treatment.

ENaC channel activity is regulated by a number of mechanisms, including aldosterone-stimulated intracellular trafficking of the ENaC -, -, and -subunits from the cytoplasm to the apical plasma membrane ( 29, 31, 35 ). Because the synthesis of -ENaC has been suggested to be a rate-limiting factor of the multimeric ENaC complex ( 33 ), sodium transport could be expected to be proportional to the abundance of the -ENaC protein levels. Recent studies have documented, however, that -ENaC and -ENaC are also important factors in the regulation of sodium transport. First, studies of ENaC subunit expression in X. laevis oocytes demonstrated that elimination of either -ENaC or -ENaC reduced sodium transport to 10% of that when all three subunits were expressed ( 9 ), and coexpression of -ENaC and ENaC with ENaC in Xenopus oocytes increased plasma membrane expression about threefold ( 4 ). Second, -ENaC- or -ENaC-deficient mice have very high urinary sodium excretion, low urinary potassium excretion, severe hyperkalemia, and die before adulthood, indicating that -ENaC and -ENaC are essential for sodium reabsorption in the distal nephron ( 6, 34 ). Moreover, the importance of the -ENaC or -ENaC subunits in volume regulation has been emphasized in studies that have identified mutations in -ENaC or -ENaC as the basis of the pathogenesis of Liddle's syndrome, a disorder characterized by volume expansion and hypertension ( 21, 41 ). Therefore, downregulation of any of the ENaC subunits would be predicted to have a severe impact on collecting duct sodium reabsorption and regulation of extracellular fluid volume. Thus the selective downregulation of -ENaC and -ENaC in lithium-induced NDI is likely to play a significant role in renal sodium wasting.

Evidence for impaired aldosterone and vasopressin regulation of ENaC subunits in lithium-treated rats. In the present study, we demonstrate physiological and biochemical changes that are compatible with impaired ENaC regulation by aldosterone and vasopressin in the cortex and outer medulla of lithium-treated rats. Physiological analysis demonstrated that lithium treatment was associated with increased fractional excretion of sodium despite increased plasma aldosterone concentration, decreased glomerular filtration rate, and a decreased filtered load of sodium. It has long been recognized that circulating levels of aldosterone regulate renal sodium reabsorption. The increased plasma aldosterone concentration was possibly caused by sodium depletion and extracellular fluid volume contraction in lithium-treated rats. The observed increase in the fractional excretion of sodium indicated that 1 ) lithium treatment is associated with impairment of the tubular reabsorption of filtered sodium and 2 ) increased plasma aldosterone was ineffective in increasing sodium reabsorption in the aldosterone-responsive renal tubule segments for maintaining sodium balance. In support of this are findings by Thomsen et al. ( 46, 48 ) demonstrating an inability of aldosterone to decrease the consumption of hypertonic NaCl in lithium-treated adrenalectomized rats and no significant effect of aldosterone or amiloride on sodium metabolism in concious, catheterized (venous, arterial, and urinary bladder) lithium-treated rats with computer-controlled servo replacement of urinary water and sodium loss. Moreover, the lithium-treated rats were severely polyuric with low urine osmolality consistent with decreased vasopressin effects.

Biochemically, the impaired aldosterone response was evidenced by unchanged -ENaC expression in the cortex and outer medulla and decreased NCC expression in the cortex, both of which are normally strongly induced by increases in plasma aldosterone seen in response to lithium treatment ( 25, 31 ). The mechanism for the possible lithium interaction with the aldosterone-signaling pathway is not known. The lithium-induced inhibition of adenylyl cyclase ( 12, 13 ) is also likely to play a role in the lack of vasopressin effects on ENaC in the cortex and outer medulla, as evidenced by markedly reduced -ENaC and -ENaC protein abundances, as well as decreased AQP2 expression. All three of these proteins are normally strongly upregulated by increases in vasopressin levels such as those induced by lithium treatment ( 18 ). Immunocytochemical analysis of ENaC subunits showing decreased labeling in the cortical and outer medullary collecting duct confirmed a dysregulation of - and -ENaC subunits independent of sodium intake (Figs. 3 and 7 ).' ]' Q- \$ v+ V3 q7 X

In contrast, ENaC regulation in the connecting tubule and inner medullary collecting duct in rats with a fixed sodium intake was consistent with normal actions mediated by increased plasma aldosterone and vasopressin concentrations. The increase in -ENaC and the 70-kDa form of -ENaC is consistent with the effects of aldosterone ( 31 ). The increase in -ENaC (sum of both 85- and 70-kDa forms) in the inner medulla is consistent with an effect of vasopressin ( 15 ). Furthermore, there was increased apical labeling of ENaC subunits in both the connecting tubule and inner medullary collecting duct, consistent with redistribution of ENaC mediated by elevated aldosterone concentrations. In contrast, no apparent apical labeling of ENaC subunits was seen in lithium-treated rats with free access to sodium (more sodium intake). Thus the redistribution of ENaC in the connecting tubule and inner medullary collecting duct was dependent on sodium intake, whereas plasma lithium was not different, therefore likely to be compensating for reduced sodium reabsorption in the cortical collecting duct and outer medullary collecting duct. In addition, the increased 1 -subunit of the Na-K-ATPase in the inner medulla is supporting evidence of a compensatory effect to prevent urinary sodium loss. Thus lithium-induced impaired aldosterone- and vasopressin-mediated regulation of ENaC is specific to the cortical and outer medullary colleting duct and is likely to play an important role in the development of natriuresis and decreased urinary concentration ability in rats with lithium-induced NDI. It remains unknown whether the putative compensatory changes in the connecting tubule and inner medulla represent direct effects of aldosterone and vasopressin or whether other signaling cascades insenstive to lithium and unique to the connecting tubule and inner medulla are involved. Interestingly, we have recently shown that ENaC redistirubtion in the connecting tubule was not sensitive to blockade of the mineralocorticoid receptor by spironolactone ( 35 ), supporting the possibility of alternative signaling pathways for ENaC trafficking. This could potentially include angiotensin II, because it was recently demonstrated by Beutler et al. ( 7 ) that treatment with candesartan (an AT 1 -receptor antagonist) reduced -ENaC expression in the presence of spironolactone (an aldosterone-receptor blocker). In addition, Christensen et al. ( 11 ) have recently shown distinct differences in the trafficking of AQP2 along the connecting tubule and collecting duct subsegments, providing further evidence of axial heterogeneity of cells expressing ENaC and AQP2.2 m0 s  P- B, o) L+ e; N9 i$ F

Future studies will be important in defining the mechanism that governs the selective and segment-specific downregulation of -ENaC and -ENaC in lithium-induced NDI. One may speculate that there could be differences in the luminal lithium concentrations along the connecting tubule and collecting duct subsegments and that this potentially may play a role. Also, the exit pathway for lithium across the basolateral membrane may be important in the determination of intracellular lithium concentration and transepithelial lithium reabsorption.7 g! I8 U0 h# m; L5 Z# s

Decreased abundance of NCC in lithium-treated rats. In addition to ENaC regulation, we demonstrated that NCC in the distal convoluted tubule was significantly downregulated, which may also contribute to the sodium-losing state seen with chronic lithium administration. This is consistent with impaired aldosterone responsiveness because aldosterone is known to regulate NCC expression ( 1, 25, 35 ). However, NCC expression is also regulated by other mechanisms, including sodium delivery to the distal convoluted tubule ( 1, 10, 42 ), which may be decreased in lithium-treated rats due to the decreased glomerular filtration rate.% j7 {( b( F% r

Summary. In this study, we have identified the renal tubule segments and the ENaC subunits involved in increased urinary sodium excretion in response to chronic lithium treatment. This involves dysregulation of -ENaC and -ENaC in the cortical and outer medullary collecting duct. The results suggest a reduced responsiveness to aldosterone and vasopressin in these specific renal tubule segments. The dysregulation of -ENaC and -ENaC is likely to play an important role in the development of natriuresis and partly in the decreased urinary concentrating ability in rats with lithium-induced NDI., Z% n2 E# M5 Y# C; H

DISCLOSURES
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The Water and Salt Research Center at the University of Aarhus is established and supported by the Danish National Research Foundation (Danmarks Grundforskningsfond). Support for this study was provided by the Karen Elise Jensen Foundation, Human Frontier Science Program, Novo Nordic Foundation, Danish Medical Research Council, University of Aarhus Research Foundation, the University Aarhus, the European Commission (QRLT 2000 00778 and QRLT 2000 00987), and the intramural budget of the National Heart, Lung, and Blood Institute.

ACKNOWLEDGMENTS( \- }. r$ _- b/ z# Z

The authors thank Lotte Vallentin Holbech, Ida Maria Jalk, Gitte Kall, Inger Merete Paulsen, Helle Høyer, Zhila Nikrozi, Mette Vistisen, and Dorte Wulff for technical assistance. The authors also thank Annette Nielsen and Dr. Thomas Jonassen at the Department of Pharmacology, University of Copenhagen, for kindly providing measurements of lithium concentration.8 `! c5 [. B  x9 q3 A* D
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