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Jakob Nielsen, Tae-Hwan Kwon, Jrgen Frkir, Mark A. Knepper and Sren Nielsen
Am J Physiol Renal Physiol 290:F1222-F1233, 2006. First published 6 December 2005; doi:10.1152/ajprenal.00321.2005 You might find this additional info useful. This article cites 55 articles, 35 of which can be accessed free at: http://ajprenal.physiology.org/content/290/5/F1222.full.html#ref-list-1
Lithium-induced NDI in rats is associated with loss of -ENaC regulation by aldosterone in CCD
This article has been cited by 5 other HighWire hosted articles Aldosterone-mediated apical targeting of ENaC subunits is blunted in rats with streptozotocin-induced diabetes mellitus Heidi O'Neill, Janne Lebeck, Patrick B. Collins, Tae-Hwan Kwon, Jrgen Frkir and Sren Nielsen Nephrol. Dial. Transplant., May , 2008; 23 (5): 1546-1555. [Abstract] [Full Text] [PDF]
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Amiloride restores renal medullary osmolytes in lithium-induced nephrogenic diabetes insipidus Jennifer J. Bedford, John P. Leader, Rena Jing, Logan J. Walker, Janet D. Klein, Jeff M. Sands and Robert J. Walker Am J Physiol Renal Physiol, April 1, 2008; 294 (4): F812-F820. [Abstract] [Full Text] [PDF] Aldosterone-Mediated Apical Targeting of ENaC Subunits is Blunted in Rats with Streptozotocin-Induced Diabetes Mellitus Heidi ONeill, Janne Lebeck, Patrick B. Collins, Tae-Hwan Kwon, Jrgen Frkir and Sren Nielsen Nephrol. Dial. Transplant., November 19, 2007;. [PDF] Functional cross talk between ENaC and pendrin Rebecca P. Hughey and Thomas R. Kleyman Am J Physiol Renal Physiol, November 1, 2007; 293 (5): F1439-F1440. [Full Text] [PDF] Cell biological aspects of the vasopressin type-2 receptor and aquaporin 2 water channel in nephrogenic diabetes insipidus Joris H. Robben, Nine V. A. M. Knoers and Peter M. T. Deen Am J Physiol Renal Physiol, August 1, 2006; 291 (2): F257-F270. [Abstract] [Full Text] [PDF] Updated information and services including high resolution figures, can be found at: http://ajprenal.physiology.org/content/290/5/F1222.full.html Additional material and information about AJP - Renal Physiology can be found at: http://www.the-aps.org/publications/ajprenal
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AJP - Renal Physiology publishes original manuscripts on a broad range of subjects relating to the kidney, urinary tract, and their respective cells and vasculature, as well as to the control of body fluid volume and composition. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2006 by the American Physiological Society. ISSN: 0363-6127, ESSN: 1522-1466. Visit our website at http://www.the-aps.org/.
Am J Physiol Renal Physiol 290: F1222F1233, 2006. First published December 6, 2005; doi:10.1152/ajprenal.00321.2005.
Jakob Nielsen,1,2 Tae-Hwan Kwon,1,3 Jrgen Frkir,1,4 Mark A. Knepper,5 and Sren Nielsen1,2
The Water and Salt Research Center and 2Institute of Anatomy, University of Aarhus, Aarhus, Denmark; 3Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea; 4Institute of Clinical Medicine, University of Aarhus, Aarhus, Denmark; and 5Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
Submitted 8 August 2005; accepted in nal form 26 November 2005
Nielsen, Jakob, Tae-Hwan Kwon, Jrgen Frkir, Mark A. Knepper, and Sren Nielsen. Lithium-induced NDI in rats is associated with loss of -ENaC regulation by aldosterone in CCD. Am J Physiol Renal Physiol 290: F1222F1233, 2006. First published December 6, 2005; doi:10.1152/ajprenal.00321.2005.Lithium-induced nephrogenic diabetes insipidus (Li-NDI) is associated with increased urinary sodium excretion and decreased responsiveness to aldosterone and vasopressin. Dysregulation of the epithelial sodium channel (ENaC) is thought to play an important role in renal sodium wasting. The effect of 7-day aldosterone and spironolactone treatment on regulation of ENaC in rat kidney cortex was investigated in rats with 3 wk of Li-NDI. Aldosterone treatment of rats with Li-NDI decreased fractional excretion of sodium (0.83 0.02), whereas spironolactone did not change fractional excretion of sodium (1.10 0.11) compared with rats treated with lithium alone (1.11 0.05). Plasma lithium concentration was decreased by aldosterone (0.31 0.03 mmol/l) but unchanged with spironolactone (0.84 0.18 mmol/l) compared with rats treated with lithium alone (0.54 0.04 mmol/l). Immunoblotting showed increased protein expression of -ENaC, the 70-kDa form of -ENaC, and the Na-Cl cotransporter (NCC) in kidney cortex in aldosterone-treated rats, whereas spironolactone decreased -ENaC and NCC compared with control rats treated with lithium alone. Immunohistochemistry conrmed increased expression of -ENaC in the late distal convoluted tubule and connecting tubule and also revealed increased apical targeting of all three ENaC subunits (, , and ) in aldosterone-treated rats compared with rats treated with lithium alone. Aldosterone did not, however, affect -ENaC expression in the cortical collecting duct (CCD), which showed weak and dispersed labeling similar to that in rats treated with lithium alone. Spironolactone did not affect ENaC targeting compared with rats treated with lithium alone. This study shows a segment specic lack of aldosterone-mediated -ENaC regulation in the CCD affecting both -ENaC protein expression and trafcking, which may explain the increased sodium wasting associated with chronic lithium treatment. epithelial sodium channel; cortical collecting duct; hypertension; nephrogenic diabetes insipidus
CHRONIC LITHIUM TREATMENT is commonly used in the management of patients with bipolar affective disorders (53). Lithium treatment is, however, often complicated by side effects including polyuria, urinary concentrating defect (3, 13, 26, 30), and increased urinary sodium excretion (4, 5, 41, 49, 51, 52). The molecular mechanism causing lithium-induced sodium loss is not well understood. Lithium is excreted mainly by the kidney (37). In the kidney tubule, lithium is reabsorbed in
competition with sodium by the type 3 Na/H exchanger (NHE3), type 1 bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), and the epithelial sodium channel (ENaC) (18, 20, 37). Approximately 60% of the ltered lithium load is reabsorbed in the proximal tubule, and 20% is reabsorbed in the thick ascending limb, connecting tubule (CNT), and cortical collecting duct (CCD) (37). During conditions with extracellular uid volume contraction and activation of the reninangiotensin-aldosterone system, increased sodium reabsorption also leads to increased lithium reabsorption. Thus daily sodium intake modulates renal lithium clearance (43), and insufcient dietary sodium intake can lead to fatal lithium intoxication in patients. Understanding of the cause of the sodium loss is therefore clinically important. The underlying mechanism is thought to involve dysregulation of ENaC through a decreased responsiveness to vasopressin and aldosterone in the CCD (4, 36, 50, 52). ENaC consists of three homologous subunits, -, -, and -ENaC (9). ENaC is the main site of sodium transport across the apical plasma membrane in the CNT and CCD (19), where it reabsorbs a large fraction of the sodium delivered from the distal convoluted tubule (DCT) (1, 46). ENaC regulation is complex and includes changes in protein expression of the individual subunits (14, 31), redistribution of ENaC subunits to and from the apical plasma membrane (28, 31), and changes in channel open probability (2, 10 12, 21, 39, 40, 54, 55). Vasopressin is known to increase protein expression of the - and -ENaC subunits, whereas aldosterone increases protein expression of -ENaC (14, 29, 31). Redistribution of ENaC-containing vesicles to the apical plasma membrane is induced by both vasopressin (7, 8, 33, 45) and aldosterone (29, 31, 38). In a previous study (36), we showed a segmentspecic downregulation of the vasopressin-regulated - and -ENaC subunits in the CCD and outer medulla but not in the CNT in rats with lithium-induced nephrogenic diabetes insipidus (NDI), in which plasma aldosterone and vasopressin levels are known to be increased (17). Furthermore, ENaC was only expressed in the apical plasma membrane domain in the CNT, not in the CCD, despite elevated plasma aldosterone. The decreased apical ENaC expression in the CCD is consistent with other previous studies (4, 50, 52) demonstrating decreased amiloride-sensitive sodium transport in chronically lithium-treated rats, suggesting a decreased aldosterone responsiveness.
290 MAY 2006
etry values to facilitate comparisons. Results are listed as the relative band densities between the groups and are not absolute, hence the term semiquantitative immunoblotting. Immunohistochemistry. The tissue was dehydrated in graded ethanol and left overnight in xylene. After tissue embedding in parafn, 2-m sections were cut on a rotary microtome (Leica Microsystems, Herlev, Denmark). For immunolabeling, the sections were dewaxed with xylene and rehydrated with graded ethanol. Sections had endogenous peroxidase activity blocked with 0.5% H2O2 in absolute methanol for 10 min. With the use of a microwave oven, the sections were boiled in a target retrieval solution (1 mmol/l Tris, pH 9.0, with 0.5 mmol/l EGTA) for 10 min. After cooling, nonspecic binding was blocked with 50 mmol/l NH4Cl in PBS for 30 min followed by 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 4C. The sections were washed min with PBS wash buffer containing 0.1% BSA, 0.05% saponin, and 0.2% gelatin and incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit immunoglobulin, DAKO P448; DAKO) for 1 h at room temperature. After 3 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 (Kem-en Tek, Copenhagen, Denmark) dissolved in distilled water with 0.1% H2O2. Mayers hematoxylin was used for counterstaining, and after dehydration, coverslips were mounted with hydrophobic medium (Eukitt; O. Kindler, Freiburg, Germany). For sections prepared for immunouorescence, a secondary uorescent 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 1 h of incubation at room temperature, coverslips were mounted with a hydrophilic mounting medium containing antifading reagent (n-propyl-gallat, P-3101; Sigma Chemical). Light microscopy was carried out with a Leica DMRE microscope (Leica Microsystems). Laser confocal microscopy was carried out on a Leica TCS-SP2 laser confocal microscope (Heidelberg, Germany). Antibodies. Rabbit polyclonal antibodies to the following renal sodium transporters were utilized: NHE3 of the proximal tubule (16),
NKCC2 of the thick ascending limb (23), the thiazide-sensitive Na-Cl cotransporter (NCC) of the DCT (25), the ENaC -, -, and -subunits in the CNT and CCD (31, 34), the monoclonal 1-subunit of Na-K-ATPase in all renal tubular segments (34), and calbindin-D28k (47). The polycolonal antisera were afnity puried against the immunizing peptides as previously described (23, 25). Specicity of the antibodies has been demonstrated by showing unique peptideablatable bands on immunoblots and specic labeling by immunocytochemistry. Presentation of data and statistical analyses. Quantitative data are presented as mean SE. Data were analyzed by one-way ANOVA followed by Bonferronis multiple-comparisons test. Multiple-comparisons tests were only applied when a signicant difference was determined in the ANOVA (P 0.05). P values 0.05 were considered statistically signicant.
Aldosterone infusion increased sodium reabsorption in rats with lithium-induced NDI. To study the direct effect of aldosterone on renal sodium handling under lithium-induced NDI, we rst treated rats with lithium for 20 days to induce polyuria. Rats with lithium-induced NDI and control rats received a relatively high amount of sodium (3.4 mmol Nadayg body wt1) during the experimental period to suppress the endogenous plasma aldosterone level in rats with lithiuminduced NDI to a level similar to that in the control rats (1.1 0.3 vs. 0.6 0.2 nmol/l, no signicant difference; Table 1). After 20 days of lithium-treatment, all rats developed polyuria (ml/day, n 19) compared with untreated control rats (ml/day, n 4, P 0.05). The rats with lithiuminduced NDI were subsequently randomized into three intervention groups: 1) oral lithium treatment for an additional 7 days, 2) oral lithium treatment plus subcutaneous aldosterone infusion for an additional 7 days, or 3) oral lithium and spironolactone treatment for an additional 7 days. The rats
Table 1. Physiological data
Li Li Aldosterone Li Spironolactone Control
Number of rats Body wt, g Food, g/200 g body wt Li intake, g/200 g body wt Urine output, ml/24 h Plasma Osmolality, mosmol/kgH2O Na, mmol/l K, mmol/l Urea, mmol/l Creatine, mmol/l Li, mmol/l Aldosterone, nmol/l Urine Osmolality, mosmol/kgH2O Na, mmol/l K, mmol/l Urea, mmol/l Creatine, mmol/l Li, mmol/l UNaV, mmol/24 h Creatine clearance, mlmin1kg1 (UNa/PNa)/(UCreat/PCreat)
17.10.4 0.770.4.00.1 5.20.4 25.90.6 0.540.04 1.10.13617 0.510.05 6.280.75 3.520.14 6.060.14 1.110.05
15.90.4 0.730.04 2129* 3012 1461* 3.10.2* 3.50.2* 23.20.9* 0.320.03* 7.50.4* 1125* 121* 191* 502* 0.220.01* 2.440.11* 3.320.17 7.410.44* 0.830.02*
6 2345* 15.10.6* 0.680.03* 6310* 4.70.4 7.11.0 29.31.6* 0.840.18 4.11.3* 0.850.18 8.131.68 2.740.10* 5.370.30* 1.100.11
188.8.131.52.1 4.20.3 23.70.3 0.60.836158 4.541.34 3.230.09 5.840.43 1.060.14
Values are means SE. UNaV, urinary sodium excretion; (UNa/PNa)/(UCreatine/PCreatine), fractional excretion of sodium. *P 0.05, Li vs. Li aldosterone and Li vs. Li spironolactone. AJP-Renal Physiol VOL
treated with lithium alone maintained a steady polyuria and a decreased urinary concentration compared with untreated control rats [Table 1; portions of these physiological data have also been presented in a parallel study (35)]. The daily urinary sodium excretion was similar between aldosterone-treated rats and rats treated with lithium alone, whereas the spironolactonetreated rats showed a marginal decrease that, at least in part, can be explained by a small decrease in the daily food intake by spironolactone-treated rats (Table 1). The aldosteronetreated rats had a signicantly decreased (UNa/PNa)/(UCreatine/ PCreatine) (an index of fractional sodium excretion) compared with rats treated with lithium alone (Table 1), suggesting an increased sodium reabsorption induced by aldosterone treatment. Spironolactone did not change (UNa/PNa)/(UCreatine/PCreatine). The renal creatinine clearance (Ccr) was increased in aldosteronetreated rats and decreased in spironolactone-treated rats compared with rats treated with lithium alone (Table 1). Interestingly, there was a large increase in urine production in aldosterone-treated rats with lithium-induced NDI compared with rats treated with lithium alone, whereas spironolactone treatment caused a decreased urine production compared with rats treated with lithium alone (Table 1). The increased polyuria induced by aldosterone treatment was associated with a decreased plasma lithium concentration compared with rats treated with lithium alone, whereas spironolactone treatment did not change plasma lithium concentration (Table 1). Aldosterone signicantly increased protein expression of -ENaC in renal cortex of rats with lithium-induced NDI. Under normal conditions aldosterone is an important regulator of -ENaC protein expression, but in our previous study (36),
we showed that -ENaC was not increased in kidney cortex of rats with lithium-induced NDI despite signicantly elevated plasma aldosterone. In this study, to determine whether this is due to decreased aldosterone responsiveness, we therefore carried out semiquantitative immunoblotting on protein samples prepared from cortex plus the outer stripe of the outer medulla. Immunoblots showed a markedly increased -ENaC protein expression in response to aldosterone treatment in rats with lithium-induced NDI compared with rats treated with lithium alone, whereas expression was decreased in response to spironolactone treatment compared with rats treated with lithium alone (Fig. 1A). A summary of densitometric analysis of immunoblots is shown in Table 2. The total protein expression of -ENaC was unchanged between groups, but aldosteronetreated rats showed a partial molecular mass shift from an 85-kDa band to a 70-kDa band (Fig. 1C). This is a known aldosterone effect thought to represent a proteolytic cleavage of the 85-kDa -ENaC form (31). The protein abundance of the -ENaC subunit was unchanged in both aldosterone- and spironolactone-treated rats compared with rats treated with lithium alone (Fig. 1B). Aldosterone did not upregulate -ENaC protein expression or induce ENaC trafcking in the CCD in rats with lithiuminduced NDI. We have previously shown that lithium-induced NDI specically affected ENaC regulation in the CCD. To investigate the direct effect of aldosterone infusion on ENaC trafcking in the kidney cortex tubule segments expressing ENaC (i.e., late DCT, CNT, and CCD) in rats with lithiuminduced NDI, we examined tissue sections stained for the ENaC subunits. Immunolabeling of -ENaC in the CCD prin-
Fig. 1. Semiquantitative immunoblots for protein prepared from cortex plus the outer stripe of the outer medulla (OSOM). Immunoblots were reacted with epithelial sodium channel (ENaC) subunits -ENaC (A), -ENaC (B), and -ENaC (C). The equality of protein amount loaded was assured by Coomassie blue staining of the gel after electrophoresis (not shown). A: the -ENaC band appeared at 85 kDa and was increased in aldosterone-treated rats with lithium-induced nephrogenic diabetes insipidus (NDI; LiAldo rats) but decreased in spironolactone-treated rats with lithium-induced NDI (LiSpiro rats) compared with rats treated with lithium alone (Li rats). B: the -ENaC band seen at 85 kDa was unchanged between groups. C: -ENaC was seen as a narrow band around 85 kDa and a broader band around 70 kDa. The 85-kDa band was decreased but the 70-kDa band was increased in the LiAldo rats compared with Li rats. In the LiSpiro rats, the 85- and 70-kDa bands were unchanged compared with Li rats. *P 0.05 compared with Li rats.
Table 2. Densitometric analysis of ENaC immunoblots
Li Li Aldosterone Li Spironolactone
Number of rats CortexOSOM NHE3 NKCC2 NCC -ENaC -ENaC -ENaC total -ENaC 85kDa -ENaC 70kDa Na-KATPase 1 ISOM NHE3 NKCC2 Na-K-ATPase 1 IM Na-K-ATPase 1
5 1.000.09 1.000.04 1.000.09 1.000.13 1.000.09 1.000.10 1.000.17 1.000.26 1.000.04 1.000.11 1.000.06 1.000.03 1.000.15
5 0.950.05 0.850.10 1.880.13* 3.120.15* 1.170.10 0.910.11 0.500.05* 6.290.77* 1.180.08 1.270.12 1.090.08 0.920.04 0.810.14
5 0.710.05* 0.730.04* 0.640.06* 0.570.07* 1.010.08 1.030.01 1.100.09 1.100.20 0.630.06* 0.600.08* 1.000.04 0.460.10* 0.590.16
Values are means SE. NHE3, type 3 Na/H exchanger; NKCC2, Na-K-2 Cl cotransporter type 2; NCC, Na-Cl cotransporter; ENaC, epithelial sodium channel; OSOM, outer stripe of outer medulla. *P 0.05, Li vs. Li aldosterone and Li vs. Li spironolactone.
cipal cells was very weak in all three groups of lithium-treated rats compared with the untreated control rats (Fig. 2, AC vs. D). Moreover, the labeling intensity in aldosterone-treated rats did not appear different from that in rats treated with lithium alone, and the immunolabeling of the apical plasma membrane was not increased (Fig. 2, A and B). The labeling intensity of -ENaC in the spironolactone-treated rats appeared marginally decreased compared with the rats treated with lithium alone (Fig. 2, A and C). Consistent with previous ndings, the expression of -ENaC (not shown) and -ENaC was markedly reduced in the CCD of the lithium-treated rats compared with untreated control rats, and neither aldosterone nor spironolactone affected the labeling pattern in the lithium-treated rats (Fig. 2, EG vs. H). Thus there was no evidence of aldosterone-induced increase of -ENaC protein expression (in CCD) in contrast to the immunoblotting results (in kidney cortex outer stripe of outer medulla), and there was no evidence of increased apical targeting of ENaC subunits in the CCD in aldosterone-treated rats with lithium-induced NDI. Aldosterone caused distinct redistribution of ENaC subunits to the apical cell domain in the DCT and the CNT and increased -ENaC expression. In contrast to the ndings in the CCD, there was stronger immunolabeling of -ENaC at the apical plasma membrane domain and weaker cytoplasmic labeling in the CNT cells in aldosterone-treated rats with lithiuminduced NDI (Fig. 3F) compared with both the CCD in the same animal (Fig. 2B) and the CNT in rats treated with lithium alone (Fig. 3E). The rats treated with lithium alone showed only faint and dispersed cytoplasmic immunolabeling (Fig. 3E), and the spironolactone-treated rats (Fig. 3G) showed weak labeling, slightly fainter than that in rats treated with lithium alone. The increased labeling intensity and apical targeting of -ENaC in the aldosterone-treated rats was not limited to the CNT but also was observed in the late DCT (Fig. 3B). The DCT showed markedly increased apical and cytoplasmic labeling compared with rats treated with lithium alone (Fig. 3A), indicating a normal response to aldosterone in OCT and CNT.
The labeling intensity of -ENaC in the late DCT of spironolactone-treated rats appeared decreased (Fig. 3C) compared with the rats treated with lithium alone and control rats (Fig. 3, A and D), suggesting a decreased mineralocorticoid receptor activation. To conrm that the increased apical immunolabeling was restricted to the CNTs, we examined tissue sections labeled with both -ENaC (shown in green) and the CNT/DCT marker protein calbindin-D28k (shown in red) (27, 47) by using confocal laser scanning microscopy. The tubule segments with increased apical -ENaC in aldosterone-treated rats (Fig. 3J) were colabeled with calbindin-D28k (Fig. 3J, inset). The -ENaC labeling in calbindin-D28k-positive tubules (Fig. 3, I, K, and L, insets) from the other experimental groups was weak and seemed dispersed in the cytoplasm without distinct apical labeling (Fig. 3, I, K, and L). Immunolabeling with -ENaC and -ENaC in the CNT cells was dispersed throughout the cytoplasm in rats treated with lithium alone (Fig. 4, A and E), whereas aldosterone-treated rats showed markedly increased labeling in the apical plasma membrane domain and decreased immunolabeling intensity in the cytoplasm (Fig. 4, B and F) consistent with redistribution of the ENaC complex induced by aldosterone (31). The spironolactone-treated rats and control rats showed dispersed labeling in the cytoplasm with no distinct apical labeling, similar to rats treated with lithium alone (Fig. 4, C, D, H, and G). The immunolabeling pattern for -ENaC and -ENaC in the late DCT was not markedly different from that in the CNT (not shown). The tubule identity was conrmed by double-labeling of -ENaC and calbindin-D28k. The tubule segments with increased apical -ENaC labeling in aldosterone-treated rats (Fig. 4J) were colabeled with calbindin-D28k (Fig. 4J, inset). The other groups showed dispersed cytoplasmic labeling without distinct apical labeling calbindin-D28k-positive cells (Fig. 4, I, K, and L, insets). Thus the labeling pattern was similar to what was observed with immunoperoxidase labeling. Interestingly, we also found increased apical labeling of -ENaC in some, but not all, of the initial CCDs of aldosterone-treated rats compared with rats treated with lithium alone (not shown). Consistent with previous results (36), we did not detect any -ENaC labeling more distal in the CCD in any of the lithium-treated rats (not shown). Increased expression of NCC in response to aldosterone in the DCT of rats with lithium-induced NDI. In a previous study, the sodium-chloride cotransporter NCC, a known aldosterone target expressed in the DCT (25), was not increased in lithiumtreated rats despite an increase in plasma aldosterone compared with untreated control rats, suggesting decreased responsiveness to aldosterone. To test this nding directly, we examined the protein expression of NCC in the kidney cortex of the aldosterone-treated rats with lithium-induced NDI. Aldosterone induced a signicantly increased protein expression of NCC in aldosterone-treated rats and decreased expression in the spironolactone-treated rats compared with rats treated with lithium alone (Fig. 5C). Spironolactone treatment decreased protein expression of NHE3, NKCC2, and Na-K-ATPase 1-subunit in rats with lithium-induced NDI. The aldosterone and spironolactone treatments were associated with signicant changes in creatinine clearance and thus tubular sodium load. We therefore examined the protein expression of sodium transporters exwww.ajprenal.org
Fig. 5. Semiquantitative immunoblots for protein prepared from cortex and OSOM. Immunoblots were reacted with type 3 Na-H exchanger (NHE3; A), Na-K-2CL cotransporter (NKCC2; B), Na-Cl cotransporter (NCC; C), and Na-K-ATPase 1-subunit (D). A and B: NHE3 and NKCC2 were unchanged in LiAldo rats but decreased in LiSpiro rats compared with Li rats. C: NCC was increased in LiAldo rats and decreased in LiSpiro rats compared with Li rats. D: Na-K-ATPase 1 was unchanged in LiAldo rats but decreased in LiSpiro rats compared with Li rats. *P 0.05 compared with Li rats.
chronically lithium-treated rats therefore could be related to decreased capacity to reabsorb sodium in segments expressing ENaC. Since our results suggest that the late DCT and CNT are responding normally, the dysregulation of ENaC in the CCD therefore appears to play an important role in sodium wasting in the condition of lithium-induced NDI. NCC protein expression was increased with aldosterone infusion. The Na-Cl cotransporter NCC (also known as the thiazide-sensitive Na-Cl cotransporter) is expressed in the DCT and is regulated by aldosterone (25). Previously NCC was found to be unchanged or decreased with lithium treatment compared with untreated control rats, suggesting a decreased responsiveness to aldosterone (24, 26, 36). In the present study, the NCC protein expression was increased in aldosteronetreated rats and decreased in spironolactone-treated rats compared with rats treated with lithium alone. Moreover, the marked increase in the NCC is consistent with increased -ENaC expression observed in the late DCT, suggesting that aldosterone action is preserved in this tubular segment.
Spironolactone treatment caused decreased Na-K-ATPase 1-subunit, NHE3, and NKCC2 protein expression. The large decrease in Na-K-ATPase 1-subunit and NHE3 protein expression in the cortex and inner stripe of the outer medulla in the spironolactone-treated rats could be related to the decreased glomerular ltration rate observed in these rats. Particularly, the decreased protein expression of Na-K-ATPase and NKCC2 in the inner stripe of the outer medulla suggests that there is a decreased expression in the medullary thick ascending limb, compatible with a decreased delivery of tubular uid out of the proximal tubule (15). Consistent with this nding is that NKCC2 protein expression in the cortex also decreased, compatible with a decreased NKCC2 expression in the cortical thick ascending limb. It therefore appears that spironolactone caused an extracellular uid volume contraction, resulting in decreased glomerular ltration rate and decreased distal delivery. Decreased plasma lithium concentration in aldosteronetreated rats with lithium-induced NDI. The renal handling of lithium in distal tubule segments has been extensively studied,
Fig. 6. Semiquantitative immunoblots for protein prepared from the inner stripe of the outer medulla (ISOM; AC) and the inner medulla (IM; D). Immunoblots were reacted with NHE3 (A), NKCC2 (B), and Na-KATPase 1 (C and D). A: NHE3 was unchanged in LiAldo rats and decreased in LiSpiro rats compared with Li rats. B: NKCC2 was unchanged in LiAldo and LiSpiro rats compared with Li rats. C: in the ISOM, Na-K-ATPase 1 was unchanged in LiAldo rats but decreased in LiSpiro rats compared with Li rats. D: in the IM, Na-K-ATPase 1 was unchanged in LiAldo and LiSpiro rats compared with Li rats. *P 0.05 compared with Li rats.
and one proposed hypothesis for the lithium reabsorption in sodium-depleted conditions is that the low end-tubular sodium concentration leads to reduced sodium reabsorption and hyperpolarization of the apical membrane, resulting in a condition that favors lithium reuptake (44). In our study, the increased creatinine clearance in the aldosterone-treated rats resulted in an increased tubular sodium and water load and, consequently, increased tubular sodium reabsorption to maintain steady state. This is partly accommodated by increased reabsorption in the proximal tubule through the glomerulotubular balance and load-dependent increased reabsorption in the thick ascending limb. Moreover, in the DCT, CNT, and CCD, sodium reabsorption could be enhanced by the aldosterone-regulated proteins NCC and ENaC. Therefore, the increased tubular sodium load may reduce the reabsorption of lithium and plasma lithium concentration. In conclusion, we have shown a segment-specic lack of response to aldosterone on -ENaC protein expression and intracellular ENaC redistribution in the CCD in rats with
lithium-induced NDI, whereas aldosterone-mediated ENaC regulation in the late DCT and CNT appears normal. These results extend our previous ndings of decreased expression of -ENaC and -ENaC in the CCD and further support evidence that the CCD is the principal site for lithium-induced abnormalities in renal tubular sodium handling and increased urinary sodium excretion in rats with lithium-induced NDI. The underlying mechanism as to how lithium induces the altered responsiveness to aldosterone specically in the CCD remains to be determined.
ACKNOWLEDGMENTS We thank Helle Hyer, Lotte Vallentin Holbech, Ida Maria Jalk, Inger Merete Paulsen, Zhila Nikrozi, Mette Vistisen, Gitte Kall, and Dorte Wulff for expert technical assistance. GRANTS The Water and Salt Research Center at the University of Aarhus is established and supported by the Danish National Research Foundation (Danwww.ajprenal.org
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