Article

Nielsen, J. et al. Proteomic analysis of lithium-induced nephrogenic diabetes insipidus: mechanisms for aquaporin 2 down-regulation and cellular proliferation. Proc. Natl Acad. Sci. USA 105, 3634-3639

Water and Salt Research Center, University of Aarhus, DK-8000 Aarhus C, Denmark.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2008; 105(9):3634-9. DOI: 10.1073/pnas.0800001105
Source: PubMed
ABSTRACT
Lithium is a commonly prescribed mood-stabilizing drug. However, chronic treatment with lithium induces numerous kidney-related side effects, such as dramatically reduced aquaporin 2 (AQP2) abundance, altered renal function, and structural changes. As a model system, inner medullary collecting ducts (IMCD) isolated from rats treated with lithium for either 1 or 2 weeks were subjected to differential 2D gel electrophoresis combined with mass spectrometry and bioinformatics analysis to identify (i) signaling pathways affected by lithium and (ii) unique candidate proteins for AQP2 regulation. After 1 or 2 weeks of lithium treatment, we identified 6 and 74 proteins with altered abundance compared with controls, respectively. We randomly selected 17 proteins with altered abundance caused by lithium treatment for validation by immunoblotting. Bioinformatics analysis of the data indicated that proteins involved in cell death, apoptosis, cell proliferation, and morphology are highly affected by lithium. We demonstrate that members of several signaling pathways are activated by lithium treatment, including the PKB/Akt-kinase and the mitogen-activated protein kinases (MAPK), such as extracellular regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38. Lithium treatment increased the intracellular accumulation of beta-catenin in association with increased levels of phosphorylated glycogen synthase kinase type 3beta (GSK3beta). This study provides a comprehensive analysis of the proteins affected by lithium treatment in the IMCD and, as such, provides clues to potential lithium targets in the brain.

Full-text

Available from: Robert A Fenton
Proteomic analysis of lithium-induced nephrogenic
diabetes insipidus: Mechanisms for aquaporin 2
down-regulation and cellular proliferation
Jakob Nielsen*
, Jason D. Hoffert
, Mark A. Knepper
, Peter Agre
§¶
, Søren Nielsen*
, and Robert A. Fenton*
†¶
*Water and Salt Research Center, University of Aarhus, DK-8000 Aarhus C, Denmark;
Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C,
Denmark;
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health,
Bethesda, MD 20892; and
§
Department of Cell Biology, Duke University Medical Center, Durham, NC 27710
Contributed by Peter Agre, January 1, 2008 (sent for review December 11, 2007)
Lithium is a commonly prescribed mood-stabilizing drug. However,
chronic treatment with lithium induces numerous kidney-related
side effects, such as dramatically reduced aquaporin 2 (AQP2)
abundance, altered renal function, and structural changes. As a
model system, inner medullary collecting ducts (IMCD) isolated
from rats treated with lithium for either 1 or 2 weeks were
subjected to differential 2D gel electrophoresis combined with
mass spectrometry and bioinformatics analysis to identify (i) sig-
naling pathways affected by lithium and (ii) unique candidate
proteins for AQP2 regulation. After 1 or 2 weeks of lithium
treatment, we identified 6 and 74 proteins with altered abundance
compared with controls, respectively. We randomly selected 17
proteins with altered abundance caused by lithium treatment for
validation by immunoblotting. Bioinformatics analysis of the data
indicated that proteins involved in cell death, apoptosis, cell
proliferation, and morphology are highly affected by lithium. We
demonstrate that members of several signaling pathways are
activated by lithium treatment, including the PKB/Akt-kinase and
the mitogen-activated protein kinases (MAPK), such as extracellu-
lar regulated kinase (ERK), c-Jun NH
2
-terminal kinase (JNK), and
p38. Lithium treatment increased the intracellular accumulation of
-catenin in association with increased levels of phosphorylated
glycogen synthase kinase type 3
(GSK3
). This study provides a
comprehensive analysis of the proteins affected by lithium treat-
ment in the IMCD and, as such, provides clues to potential lithium
targets in the brain.
differential gel electrophoresis (DIGE) kidney GSK3
Akt kinase
L
ithium administration is the most popular therapeutic approach
to treat bipolar disorders, with 0.1% of the population receiving
lithium (1, 2). In 50% of these patients, chronic lithium treatment
is associated with altered renal function and nephrogenic diabetes
insipidus (NDI) characterized by a defective urinary concentrating
mechanism that manifests in polyuria, increased sodium excretion,
and hypercholoremic metabolic acidosis (3). The polyuria is largely
explained by decreased abundances of the vasopressin (AVP)-
regulated aquaporin 2 and aquaporin 3 (AQP2 and AQP3) water
channels in the collecting duct (4, 5). The renal sodium loss is likely
to be caused by reduced expression of the epithelial sodium channel
(ENaC) in the cortical and outer medullary collecting duct (6, 7).
In addition, lithium induces increased expre ssion of important
acid-base transporting proteins including the H
-ATPase and the
anion exchanger type 1 (AE1) in the collecting duct (8). These
changes may be, at least in part, attributable to an increase in the
proportion of intercalated cells compared with principal cells (9),
which is associated with increased cell proliferation and apoptosis
of principal cells (10). Additionally, further ‘‘remodeling’’ of the
kidney can occur with lithium treatment, including major structural
changes such as medullary tubular cysts and tubular atrophy,
resulting in tubulointerstitial fibrosis and renal failure (11).
Despite numerous studies documenting the consequences of
chronic lithium treatment, little is known about the underlying
mechanism and signaling pathways affected by lithium. In this
study, we describe the use of differential gel electrophoresis
(DIGE) combined with matrix-assisted laser desorption-ionization
time-of-flight (MALDI-TOF) mass spectrometry (MS) and bioin-
formatics to identify proteins with altered abundance in the inner
medullary collecting ducts (IMCD) of lithium-treated rats and their
possible cellular function.
Results
Physiological Effect of Lithium Treatment. Chronic lithium adminis-
tration resulted in a profound urinary concentrating defect. In both
study 1 and study 2, water intake was significantly increased after
4 days of lithium treatment (Fig. 1A). During this time period, urine
volumes changed correspondingly and were increased 3-fold after
1 week of lithium treatment and 6-fold after 2 weeks of lithium
treatment [see Fig. 1B and supporting information (SI) Table 6].
Thus, the effects of lithium on renal water excretion were estab-
lished at 1 week and progressed over the following week, indicating
further changes in renal function. The observed polyuria was
accompanied by a time-dependent reduction in AQP2 protein
abundance in the IMCD (Fig. 1C), with AQP2 protein expression
decreased to 58% of control rats at 1 week of lithium treatment and
further decreased to 33% of control rats at 2 weeks of lithium
treatment consistent with previous studies (10).
DIGE Combined with MALDI-TOF-TOF Identified Multiple Unique Pro-
teins Regulated by Lithium.
To identify proteins in the IMCD
proteome with altered abundance in lithium-treated rats compared
with control rats, we carried out 2D DIGE followed by tandem MS.
On each of five 2D gels, the fluorescence of equivalent amounts of
Cy3-labeled IMCD protein from lithium-treated rats and Cy5-
labeled protein from control rats was detected with a laser-
fluorescence scanner. A representative gel image is shown in Fig.
2. For statistical analysis, the fluorescence signals for each protein
spot were normalized between gels, and protein spots with signif-
icantly changed signal intensities were chosen for identification (23
and 159 protein spots from weeks 1 and 2, respectively). Proteins
were considered positively identified if (i) the total protein score
(MS certainty estimate) based on the peptide fingerprint was 99%
or (ii) the total protein score based on peptide fingerprint was
95% combined with a total ion score (tandem MS certainty
Author contributions: J.N. and R.A.F. designed research; J.N., J.D.H., and R.A.F. performed
research; J.N., M.A.K., P.A., S.N., and R.A.F. analyzed data; and J.N., M.A.K., P.A., S.N., and
R.A.F. wrote the paper.
The authors declare no conflict of interest.
To whom correspondence may be addressed. E-mail: rofe@ana.au.dk or pagre@cellbio.
duke.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0800001105/DC1.
In addition, 56 protein spots that did not have significantly different expression by DIGE
analysis were chosen for identification.
© 2008 by The National Academy of Sciences of the USA
3634–3639
PNAS
March 4, 2008
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Page 1
estimate) based on peptide sequence was 95%, and (iii)the
molecular weight and the isoelectric point of the identified protein
were consistent with the protein spot location on the 2D gel. These
parameters resulted in identification of 6 and 74 different proteins
with changed expression at 1 and 2 weeks, re spectively (Tables 1
and 2).
An additional animal study, with physiological parameters ob-
served similar to study 1 (SI Table 6), was performed followed by
immunoblotting to confirm the abundance changes in several of the
proteins identified by using MALDI-TOF-TOF (Fig. 3 and SI
Table 7). The changes in protein abundance observed by immu-
noblotting showed a general correlation with the changes in protein
spot intensities obtained by using DIGE (Fig. 4).
Pathway Analysis Identified Cell Death and Apoptosis as Major
Cellular Functions Affected by Lithium.
To explore the functional
roles of the changed proteins in lithium-treated rats and to generate
hypothesis for further studies, we performed bioinformatic pathway
analysis using Ingenuity Pathway Analysis (IPA) software. Because
we were interested in identifying candidate proteins and signaling
mechanisms involved in the total cellular re sponse to lithium, both
1- and 2-week time points were included in the pathways analysis.
The IPA analysis revealed that a large fraction of the proteins we
identified were associated with cell structure (e.g., annexin 2,
annexin 5, ezrin, lamin A), cell death (e.g., adenine phosphoribo-
syltransferase, mortalin, cyclophilin A), and apoptosis (e.g., perox-
iredoxin-5, programmed cell death protein 6, retinaldehyde
dehydrogenase 3) (Table 3, SI Table 8). In addition, based on the
IPA analysis, we proposed that signaling pathways involving the
PKB/Akt-k inase and the mitogen-activated protein kinases
(MAPK)—including extracellular regulated kinase (ERK), c-Jun
NH
2
-terminal kinase (JNK), and p38 MAPK—could be involved in
mediating both the lithium-induced changes on cell viability and cell
structure as well as the reduced expression of AQP2 observed
during chronic lithium treatment.
Lithium Treatment Increases Phosphorylation of PKB (Akt) and Gly-
cogen Synthase Kinase Type 3
(GSK3
). To assess these predictions,
changes in both protein abundance and phosphorylation state of
these kinases were analyzed (Fig. 5, Table4,andSI Table 9). Total
protein abundance of Akt was unchanged at both 1 and 2 weeks of
lithium treatment compared with control rats, whereas the phos-
phorylated forms pS473Akt and pT308Akt were increased 2-fold.
After 1 week of lithium treatment, the downstream cell-cycle
controlling protein P27/Kip1 also was decreased, possibly because
of degradation via a phospho-Akt-mediated mechanism. In the
same pathway, phosphorylated (active) Akt further phosphorylates
GSK3
at serine 9 thereby inhibiting GSK3
kinase activity.
Consistent with this pathway, the abundance of phosphorylated
pS9-GSK3
was increased 2.5-fold and 4-fold at 1 and 2 weeks of
lithium treatment, respectively.
A
B
C
Fig. 1. Effects of lithium treatment on water balance and AQP2 protein
expression in study 1. (A) Daily water intake was increased in the lithium-
treated rats at day 4 and progressively increased until day 14. (B) Daily urine
volume was increased in parallel with the water intake. (C) Immunoblotting of
AQP2 in the IMCD showed decreased expression at 1 week and 2 weeks of
lithium treatment compared with untreated control rats.
*
, P 0.05.
AB
C
D
Fig. 2. Representative images of Cy3- and Cy5-labeled protein on a 2D gel.
(A) Fluorescence of Cy3-labeled IMCD protein from lithium-treated rats shown
as red pseudo-color. (B) Fluorescence of Cy5-labeled IMCD protein from
control rats shown as green pseudo-color. (C) Merged image of A and B with
box indicating magnified image in D.(D) Magnification of boxed section in C.
Protein spots with equal protein abundance appear yellow (arrow 1), and
proteins with increased or decreased protein expression in lithium-treated
rats appear red (arrow 2) and green (arrow 3), respectively.
Table 1. Proteins identified by MALDI-TOF-TOF after 1 week
of lithium treatment
Protein name Lithium/Control
78-kDa glucose-regulated protein (GRP78) 1.6
Cathepsin D 1.6 1
Fatty acid-binding protein 0.65*
Myosin light polypeptide 6 0.57
Prohibitin 1.5* 1
T-plastin (Plastin-3) 1.8 8
Annexin 5 0.98 NS 8
Carbonic anhydrase 2 1.1 NS 1
Elongation factor Tu (eEF1A-1) 1.7 NS 1
Glutathione S-transferase
1 0.79 NS 1
Glutathione S-transferase
1.07 NS 1
Heat shock 70-kDa protein 1B 0.90 NS 8
Heat shock 70-kDa protein 1-like 0.90 NS 2
Hypoxanthine-guanine phosphoribosyltransferase 1.4 NS 1
NADH dehydrogenase 0.64 NS 1
Protein disulfide-isomerase A3 (ERp60) 1.1/1.4 NS 1
Retinaldehyde dehydrogenase 3 1.3 NS 2
Superoxide dismutase Cu-Zn 0.85/1.2 NS 1
Thioredoxin domain-containing protein 4 1.1 NS 1
Transketolase 1.2 NS 1
-Actin 1.1* NS 1
Names of identified proteins and the ratio of protein abundance (lithium/
control) after 1 week of lithium treatment. Additional identified proteins with
nonsignificant change (NS) at 1 week but significant changes at 2 weeks of
lithium treatment are included in the table. The arrows indicate the change in
protein abundance observed after 2 weeks of lithium treatment. Bidirectional
arrows indicate that both up- and down-regulation were observed in differ-
ent spots. Unmarked values were identified by MS with certainty scores 99%.
Values marked with asterisks (*) were identified with certainty score 95% by
both MS and tandem MS.
Nielsen et al. PNAS
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Lithium Treatment Increases Phosphorylation of ERK, JNK, and P38.
ERK, JNK, and P38 MAPK also were affected by lithium. Al-
though phosphorylated pERK2 was increased only at 2 weeks, total
ERK2 abundance was increased at both 1 and 2 weeks (Fig. 5, Table
4). Total JNK1 and JNK2 abundance as well as (active) phosphor-
ylated pJNK1 were increased at both time points, alongside total
and phosphorylated P38 (activated) (Fig. 5, Table 4). One target of
P38 MAPK is phosphorylation of HSP27 (identified with DIGE-
MALDI-TOF-TOF). HSP27, which is important for cell survival
during cell stress (12), was increased at 1 week of lithium treatment
but markedly decreased after 2 weeks of lithium treatment (Fig. 3,
Table 2).
Lithium Treatment Results in Increased
-Catenin and E-Cadherin
Abundance. A potential target for GSK3
is
-catenin, which is
phosphorylated by GSK3
, resulting in its proteasomal degradation
Table 2. Proteins identified by MALDI-TOF-TOF after 2 weeks
of lithium treatment
Protein name Lithium/Control
14-3-3 and 14-3-3
3.2 and 0.44
26S proteasome non-ATPase regulatory subunits 8 and 13 2.7 and 1.5
3-Hydroxyacyl-CoA dehydrogenase type 2 2.9*
Aconitase 1.7
Actin-like protein 3 1.9
Adenine phosphoribosyltransferase 3.1/2.2
Adenosine kinase 2.1
Aldose 1-epimerase 0.52
Aldose reducatse 0.51*
Annexin 2 and 4 and 5 0.64 and 1.5 and 1.7*
ATP synthase
and
chain 1.7* and 2.5
Calcyclin 0.61
Calmodulin 0.62*
Carbonic anhydrase 2 2.0*
Cathepsin D 3.4
Cofilin-1 1.6
Creatine kinase B type 2.4
Cyclophilin A 1.6/1.8/2.4
Cytochrome c oxidase subunits 5A and 5B 1.6* and 2.3*
Dynein light chain 2A 4.3*
ECH1 (Q62651) 1.6
Electron transfer flavoprotein
-subunit 2.7
Elongation factor Tu (eEF1A-1) 1.8
Eukaryotic translation initiation factor 3 subunit 12 5.8
Ezrin 2.2
Ferritin light chain 1 2.5
Fructose-bisphosphate aldolase A 1.5/0.64*
Glutathione S-transferase
1 and
2.9 and 3
GTP-binding nuclear protein Ran 1.7*
Heat shock 27-kDa protein 0.5*
Heat shock 70-kDa protein 1B 0.64/1.7
Heat shock 70-kDa protein 1-like 0.64*
Hypoxanthine-guanine phosphoribosyltransferase 1.7
Lamin A 1.6/5.5
L-lactate dehydrogenase A chain 0.45/2.3
Malate dehydrogenase 0.47/1.8
Mortalin 2.4*
NADH dehydrogenase 1.9/2.2
Nerve growth factor-induced protein 42A 0.51*
Neurostimulating peptide (HCNP) 1.9/2.4
NSFL1 cofactor p47 1.5/1.9
Nucleoside diphosphate kinase B 1.6*
Oxalosuccinate decarboxylase 0.6
Peroxiredoxin-5 1.8*
Phosphoglycerate mutase isozyme B 1.7/2.7
Programmed cell death protein 6 2.1
Prohibitin 2.7*
Proteasome activator complex subunit 2 1.8/2.7/3.2*
Proteasome subunit
types 1 and 9 4.7 and 2.6*
Protein disulfide-isomerase A3 (ERp60) 2.0*
Retinaldehyde dehydrogenase 3 0.62
SNAP-25-interacting protein 1.8
Stress-induced-phosphoprotein 1 (STI1) 1.6
Superoxide dismutase Cu-Zn 1.8
Thioredoxin domain-containing protein 4 1.6
T-plastin (Plastin-3) 0.64/1.7
Transketolase 1.8
Triosephosphate isomerase 1.8
Tropomyosin-1
chain 0.44
Tumor rejection antigen gp96 1.5
Ubiquinol-cytochrome c reductase iron-sulfur subunit 1.8*
Uridine monophosphate kinase 2.7
Vacuolar protein sorting-associated protein 29 1.6*
-Crystallin B chain 0.62*
-Glucosidase 2 2.3
-Actin 1.7*
Identified proteins and the ratio of protein abundance (lithium/control)
after 2 weeks of lithium treatment. Unmarked values were identified by MS
with certainty scores 99%. Values marked with asterisks (*) were identified
with certainty score 95% by both MS and tandem MS. Multiple numbers are
the same protein identified in different spots.
Fig. 3. Confirmatory immunoblotting for IMCD proteins identified as
changed by lithium treatment. Each row shows a representative immunoblot,
and each lane is loaded with a sample from a different rat (n 6 rats per
treatment).
*
, P 0.05.
Fig. 4. Correlation of changes in protein expression determined by DIGE
analysis versus immunoblotting. The ratio of protein expression in lithium/control
as determined by immunoblotting and DIGE analysis are expressed on the x axis
and y axis, respectively. Proteins identified at both 1 and 2 weeks are included.
3636
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Page 3
(13). Thus, a decrease in active pS9-GSK3
would be predicted to
result in increased
-catenin. Indeed, after 2 weeks of lithium
treatment,
-catenin abundance is increased significantly (Fig. 5,
Table 4) compared with controls. Increased
-catenin not associ-
ated with the cell-to-cell cadherin complex can accumulate intra-
cellularly affecting the Wnt-signaling cascade. To examine this
possibility, immunolabeling of
-catenin in control or lithium-
treated rats was performed (Fig. 6). In both control rats and rats
after 1 week of lithium treatment,
-catenin labeling is localized
predominantly to the basolateral plasma membrane domain. In
contrast, in rats after 2 weeks of lithium treatment, there is a marked
increase in intracellular
-catenin labeling, suggesting accumula-
tion of
-catenin within the principal cell. Because
-catenin is a
central component of the cadherin cell-adhesion complex, the
redistribution of
-catenin may affect other proteins involved in
cell-to-cell adhesion. Indeed, the abundance of E-cadherin was
increased after lithium treatment (Fig. 5, Table 4), although this
change was not associated with altered cellular distribution (data
not shown).
Discussion
The present study uses a proteomics approach, coupled with
pathways analysis, to identify unique proteins that are involved in
the pathogenesis of lithium-induced NDI. Moreover, we examined
the activation state of a number of key cellular signaling pathways
to further investigate the cellular changes induced by lithium.
The well established lithium protocol used in our study resulted
in development of a severe polyuria within 7 days that progre ssed
in magnitude throughout the duration of the study. For the pro-
Table 3. Summary of IPA analysis
Cellular function Number of proteins
Cell death (apoptosis) 29 (22)
Cell growth and proliferation 22
Cell morphology 10
Nucleic Acid Metabolism 9
Small molecule biochemistry 23
Cell signaling 40
Carbohydrate metabolism 23
Shown are the number of identified proteins associated with molecular
pathways and cellular function as determined by IPA and ordered by decreas-
ing IPA analysis significance score.
Fig. 5. Immunoblotting for proteins predicted by pathway analysis to be
involved in lithium-induced changes. Each row shows a representative immu-
noblot, and each lane is loaded with a sample from a different rat (n 6 rats
per treatment).
*
, P 0.05.
Table 4. Summary of immunoblots of proteins predicted by
pathway analysis (IPA) to be affected by lithium
Protein name
Length of lithium treatment
1 week 2 week
Akt 109 4 110 7
p473Akt 253 16* 270 25*
p308Akt 231 30* 260 7*
P27/KIP 58 8* 117 9
GSK3
132 5* 131 4*
GSK3
136 4* 132 4*
pGSK3
258 10* 416 48*
Erk 2 133 2* 169 17*
Erk 1/2 120 10 142 13*
JNK1 295 20* 182 16*
JNK2 231 14* 179 15*
pJNK1 122 6* 149 9*
P38 268 27* 197 17*
pP38 200 10* 426 46*
-Catenin 106 2 144 2*
E-cadherin 183 10* 142 9*
Controls were normalized to 100.
*
, P 0.05 versus control at 1 week and
control at 2 weeks, respectively. Phosphorylated proteins are labeled with the
prefix p.
AB
C
D
Fig. 6. Immunoperoxidase labeling of
-catenin in the inner medulla. In
control rats (A and C),
-catenin labeling is localized predominantly to the
basolateral plasma membrane domain. After 1 week of lithium (B), labeling is
unchanged compared with control rats, but after 2 weeks (D), there is a
marked increase in intracellular
-catenin labeling within the principal cell.
(Scale bar: 20
m.)
Nielsen et al. PNAS
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PHYSIOLOGY
Page 4
teomic analysis, the two time points examined were specifically
chosen as: (i) after 7 days of lithium treatment, the proteins with
altered abundance may be involved in the mechanism that results
in down-regulation of AQP2 and the subsequent onset of polyuria;
and (ii) after 14 days of lithium treatment, proteins identified may
be important for the lithium-induced structural changes in the
kidney that occur with prolonged lithium treatment. However, it is
important to emphasize that the cellular re sponse s to lithium
treatment identified from this study could be direct responses (e.g.,
response s to increased phosphorylation of transcription factors by
‘‘lithium-regulated kinases’’) or indirect responses that are not an
immediate consequence of lithium-mediated signaling (e.g.,
changes in protein abundance attributable to the reduced intersti-
tial osmolality that occurs from prolonged polydipsia and polyuria).
The indirect effects of lithium are likely to be diverse and may
include several categorie s of response s, such as (i) induction of
transcription factors downstream of transcription factors immedi-
ately activated by lithium; (ii) secondary changes in circulating
hormone levels (such as AVP); or (iii) responses to increased
tubular flow occurring in polyuria. This study does not attempt to
discriminate between these different types of re sponses. Further-
more, several of the proteins identified in this study are key
regulators in more than one intracellular signaling process; thus, the
relative effect of lithium on each individual pathway can only be
postulated.
Proteomic analysis using stringent parameters to avoid false-
positive identifications resulted in identification of 6 and 74 differ-
ent proteins with altered abundance at 1 and 2 weeks, respectively.
Although a direct correlation was observed between the changes in
abundance from confirmatory immunoblotting and the changes in
protein spot intensities obtained by using DIGE analysis, the
magnitude of the change s between the two techniques differed
substantially (Fig. 4). This difference may be explained, in part, by
the possibility that posttranslational modifications result in proteins
migrating to several different spots on the 2D gel. For example,
after 2 weeks of lithium treatment, HSP70 was identified in two
different protein spots with decreased and increased protein ex-
pression, whereas the total protein expression was unchanged by
immunoblotting, suggesting a shift between two posttranslational
state s of HSP70 rather than a change in total abundance. Moreover,
if only one of multiple spots of a shifted protein is identified on the
DIGE gel, the protein expre ssion (based on DIGE) may appear
changed while the total protein abundance is unchanged based on
immunoblotting (e.g., CA2 or 14-3-3). Thus, the list of proteins
identified by DIGE and MALDI-TOF-TOF represents a list of
proteins with changed expression in a specific posttranslational
state. Below, we discuss some of the major regulatory targets
identified and propose their role in lithium-induced NDI.
Regulation of PKB/Akt by Lithium and Its Role in Cell Survival. The
active forms of Akt, pS473Akt and pT308Akt, were increased
2-fold after both 1 and 2 weeks of lithium treatment. Akt/PKB
acts as a critical junction in cell signaling within all cells of higher
eukaryotes. One of the key actions of Akt is to block apoptosis by
blocking the function of proapoptotic proteins (such as Bcl-2), and
promoting cell survival through activation of NF-
B signaling (14).
Chronic lithium treatment causes a multitude of structural changes
within the kidney. For example, despite increased proliferation of
principal cells, the proportion of intercalated cells to principal cells
increases after 4 weeks of lithium treatment, sugge sting that there
is a decrease in principal cell viability (9, 10). The observed increase
in Akt activation could be a survival re sponse by the principal cell
to limit apoptosis and the trigger for induced cell proliferation. In
support of this finding, lithium treatment decreases the abundance
of the p27/Kip1 cyclin-dependent kinase inhibitor, thus limiting p27
localization to the nucleus and attenuating its cell-cycle inhibitory
effects. The increased abundance of 14-3-3, which binds to p27 and
sequesters it in the cytoplasm, and of HSP27, which cause s degra-
dation of p27, adds further credence to this pathway.
Increased HSP27, which binds to Akt, is a common response to
cell stress, leading to cytoskeleton reorganization (12). This reor-
ganization of the principal cell cytoskeleton possibly could play a
direct role in the diminished AQP2 trafficking apparent after
lithium treatment (15). Additionally, Akt-dependent phosphoryla-
tion and inhibition of GSK3 (see below) is proposed to drive cell
proliferation by limiting the GSK3-mediated proteasomal degra-
dation of proteins involved in cell-cycle entry, such as G
1
cyclins and
the transcription factors c-jun and c-myc. Together, our results
suggest that Akt signaling pathways are likely responsible for the
marked principal cell proliferation observed with lithium
treatment.
Phosphorylation of GSK3
. GSK3, which is inhibited noncompeti-
tively by lithium (16, 17), has been studied extensively for its role in
cell proliferation and epithelial-mesenchymal transition (13). In our
study, lithium treatment resulted in a marked increase in phos-
phorylated (inactive) GSK3
, in line with previous studies (18).
Because GSK3
is a target for phosphorylation by Akt, the
increased p-Akt observed with lithium treatment is the potential
upstream regulator of this signaling cascade (see above). In one of
its many cellular functions, GSK3
functions as a negative regulator
of the Wnt/beta-catenin (
-catenin) pathway by phosphorylating
-catenin (see below). Overexpression of GSK3 has been shown to
make cells more sensitive to proapoptotic stimuli (19); thus, a
decrease in its activity also may enhance cell survival.
A direct role of GSK3
in lithium-induced down-regulation of
AQP2 abundance has not been demonstrated. However, it has been
shown that inhibition of GSK3
by lithium results in enhanced renal
COX2 expression in interstitial cells, leading to an increase in local
PGE(2) excretion (18), that in turn may counteract AVP actions by
causing endocytic retrieval of AQP2 from the collecting duct
plasma membrane, thus impairing urinary concentrating ability
(20). GSK3
knockout mice die from liver degeneration (21); thus,
the consequence of GSK3
gene deletion on kidney function has
yet to be examined. (A recent report has demonstrated that a
collecting duct-specific gene knockout of GSK3
results in an
impaired maximal urinary concentrating ability and a reduced
increase in AQP2 mRNA levels after water restriction.**)
Increased Intracellular Accumulation of
-Catenin.
-Catenin not
only regulates cell-to-cell adhesion as a protein interacting with
cadherin but also functions as a component of the Wnt signaling
pathway. When not assembled in complexes with cadherins,
-
catenin forms an intracellular complex with axin that is phosphor-
ylated by GSK3
, creating a signal for the rapid ubiquitin-
dependent degradation of
-catenin by proteosomes. However, if
GSK3
is inactivated, as we observed during lithium treatment,
-catenin can accumulate intracellularly and subsequently translo-
cate to the nucleus where it serves as an activator of T cell factor
(Tcf)-dependent transcription, leading to an increased expression
of several specific target genes (22). After 2 weeks of lithium
treatment, we showed a large increase in the intracellular abun-
dance of
-catenin within the IMCD. Thus, it is plausible that
lithium treatment further results in translocation of
-catenin to the
IMCD cell nucleus, where it can regulate transcription of target
genes. A previous study has reported that the effect of lithium on
AQP2 down-regulation is mediated by a transcriptional mechanism
(23). Interestingly, using bioinformatics we discovered that both the
mouse and rat AQP2 gene 5 flanking regions contain several
**Rao R, Hao C, Golovin A, Patel S, Woodgett J, Harris R, Breyer M, Collecting Duct Selective
Gene Knockout of Glycogen Synthase Kinase 3
Impairs the Renal Response to Vaso-
pressin. Renal Week 2007, October 31–November 5, 2007, San Francisco, CA, abstr.
SA-FC142.
3638
www.pnas.orgcgidoi10.1073pnas.0800001105 Nielsen et al.
Page 5
consensus sites for TCF, thus AQP2 gene transcription may be
regulated directly by TCF-dependent transcription. Alternatively,
TCF may regulate the abundance of other hierarchical transcription
factors that subsequently modulate AQP2 expression. Further-
more, TCF-dependent transcription has been demonstrated to
regulate a number of proteins involved in cell-cycle entry; thus, it
also may play a role in the principal cell proliferation observed with
lithium treatment. Direct analysis of these hypothesis using cell-
based systems will form the basis of future work.
Other Kinase-Mediated Signaling Cascades. Lithium treatment re-
sulted in either increased abundance or increased phosphorylation
of JNK, P38, and MAPK/ERK. Increased p-Akt, via the apoptosis
signal-regulating kinase 1 (ASK1), is a potential mediator of both
JNK and P38 signaling pathways. Both JNK and P38 function in
independent protein kinase cascades transducing cellular stress
signals. Additionally, P38 is centrally involved in apoptosis and
cytoskeleton reorganization after cell stress via its interaction with
HSP27 (see above).
ERK is a classical MAPK that is ubiquitously expressed and can
be activated by numerous stimuli such as growth factors. Stimula-
tion of the ERK signaling cascade modulates numerous cellular
functions, including cellular proliferation, differentiation, and sur-
vival. Interestingly, ERK inhibitors have been shown to block
AVP-induced increases in AQP2 expre ssion (24). Thus, increased
ERK activation after lithium treatment may be a cellular re sponse
to limit down-regulation of AQP2.
Conclusion
Using a stringent proteomics approach, we have identified 77
different proteins within the IMCD that are affected, either directly
or indirectly, by lithium treatment. The proteins identified have a
variety of functions, including signal transduction, regulation of
gene expre ssion, cytoskeletal organization, cellular reorganization,
apoptosis, and cell proliferation. A number of these proteins are
ubiquitously expressed, such as GSK3
, and as such also may be
involved in the actions of lithium within the brain. Our studies
clearly demonstrate that the cellular effects of lithium treatment are
broad and complex, and as such a single pathway leading to reduced
AQP2 expression and subsequent polyuria is unlikely. However, our
current study has identified numerous unique proteins that may
play a role in AQP2 regulation and thus opens up numerous
avenues of future research.
Concise Methods
Animal Protocol for Proteomics Study (Study 1) and Confirmatory Blotting
(Study 2). All animal protocols were approved by the boards of the Institute of
Anatomy and Institute of Clinical Medicine, University of Aarhus, according to the
licenses for the use of experimental animals issued by the Danish Ministry of
Justice. Male Wistar rats were housed individually in normal cages. Rats were
given daily food rations consisting of (per 200 g of body weight) 20 g of rat chow
supplemented with 1.7 mmol of NaCl (total Na intake was 3.4 mmol/200 g of body
weight) and 20 ml of tap water. Lithium-treated rats received 0.8 mmol of LiCl per
200 g of body weight. All rats had free access to water. In study 1, the number of
animals were: control, n 10; lithium-treated, n 22. In study 2, the number of
animals were: control, n 12 and lithium-treated, n 20. Half of the rats in each
study were treated for 1 week, and the other half were treated for 2 weeks. The
two lithium-treated rats with the highest urine output and the two lithium-
treated rats with the lowest urine output at the end of the study periods were
excluded from analysis. Urine was collected over 24-h periods. After 7 and 14 days
of treatment, rats were anesthetized with isoflurane, blood was collected from
the inferior vena cava, kidneys were rapidly removed, the inner medulla was
dissected, and IMCD tubules were prepared.
IMCD Tubule Preparation. The protocol for IMCD tubule suspension preparation
has been described in ref. 25.
DIGE Analysis and Protein Identification. DIGE was performed as described in
ref. 26.
Semiquantitative Immunoblotting, Immunohistochemistry, and Antibodies. Af-
ter preparation of total protein from IMCD tubules, semiquantitative immuno-
blotting was performed as described in ref. 6. Results are listed as the relative
band densities between the groups. The immunohistochemistry technique was
described extensively in ref. 6. A minimum of four control or four lithium-treated
rat kidneys were examined, and representative data are shown. Light microscopy
was carried out with a Leica DMRE microscope (Leica Microsystems). For the list of
antibodies used for immunoblotting and immunohistochemistry, including in-
formation about host animal, company, and catalogue number, see SI Table 10.
Presentation of Data and Statistical Analyses. Quantitative data are presented
as mean SE. Data were analyzed by one-way ANOVA followed by Bonferroni’s
multiple-comparisons test. Multiple-comparisons tests were applied only when a
significant difference was determined in the ANOVA (P 0.05). P values 0.05
were considered statistically significant.
ACKNOWLEDGMENTS. We thank Angel Aponte at the Proteomics Core
facility at the National Heart, Lung, and Blood Institute for expert assis-
tance. The Water and Salt Research Center at the University of Aarhus is
established and supported by the Danish National Research Foundation
(Danmarks Grundforskningsfond). J.N. was supported by the foundation of
A. P. Møller og Hustru Chastine McKinney Møllers Fond til almene Formal-
Fonden til Lægevidenskabens Fremme. R.A.F. is supported by a Marie Curie
Intra-European Fellowship and the Danish National Research Foundation.
Funding to M.A.K. was provided by the Intramural Budget of the National
Heart, Lung, and Blood Institute (National Institutes of Health Project
Z01-HL001285). Further support for this study was provided by Marie Curie
Research Training Networks (RTN) program ‘‘AQUA(GLYCERO)PORINS.’’
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Nielsen et al. PNAS
March 4, 2008
vol. 105
no. 9
3639
PHYSIOLOGY
Page 6
  • Source
    • "The exact explanation of the desensitizing effect of lithium on the renal cells has not yet been given. However, several hypothetical mechanisms have been proposed at the molecular level according to which various pathways can lead to the desensitization of the renal cells following lithium treatment (Nielsen et al., 2008). Moreover, a decrease in the number of renal cells, particularly collecting duct cells, was also observed leading to the conclusion that lithium therapy might also be directly responsible for the destruction of renal cells. "
    [Show abstract] [Hide abstract] ABSTRACT: Nephrogenic diabetes insipidus is a clinical sub-type of a diversely expounded disorder, named diabetes insipidus. It is characterized by inability of the renal cells to sense and respond to the stimulus of vasopressin. Amongst its various etiologies, one of the most inevitable causes includes lithium-induced instigation. Numerous studies reported marked histological damage to the kidneys upon long-term treatment with lithium. The recent researches have hypothesized many lithium-mediated mechanisms to explain the damage and dysfunction caused in the kidneys following lithium exposure. These mechanisms, widely, intend to justify the lithium-induced electrolyte imbalance, its interference with some vital proteins and a specific steroidal hormone, obstruction caused to a certain imperative transducer pathway and the renal tubular acidification defect produced on its prolonged therapy. Thorough study of such mechanisms aids in better understanding of the role of lithium in the pathophysiology of this disorder. Hence, the ameliorated knowledge regarding disease-pathology might prove beneficial in developing therapies that aim on disrupting the various lithium-mediated pathways. Hence, this may effectively lead to the demonstration of a novel treatment for nephrogenic diabetes insipidus, which is, at present, limited to the use of diuretics which block lithium reuptake into the body. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Mar 2015 · European Journal of Pharmacology
  • Source
    • "This initial dysregulation of vasopressin-regulated water reabsorption contributes to the urine-concentrating defect; however, long-term lithium treatment decreases the protein abundance AQP2 and UT-A1 exacerbating the effect [11]. Although lithium-dampened cAMP production is likely the primary cause of NDI, lithium dysregulation of renal prostaglandins [12], altered purinergic signaling [13] and modifications of the phosphatidylinositol signaling pathway1415161718 have also been implicated. To prevent advancing renal side effects resulting from lithium therapy, physicians may have to remove the patient from treatment regardless of its effectiveness on psychotic episodes. "
    [Show abstract] [Hide abstract] ABSTRACT: Lithium, an effective antipsychotic, induces nephrogenic diabetes insipidus (NDI) in ∼40% of patients. The decreased capacity to concentrate urine is likely due to lithium acutely disrupting the cAMP pathway and chronically reducing urea transporter (UT-A1) and water channel (AQP2) expression in the inner medulla. Targeting an alternative signaling pathway, such as PKC-mediated signaling, may be an effective method of treating lithium-induced polyuria. PKC-alpha null mice (PKCα KO) and strain-matched wild type (WT) controls were treated with lithium for 0, 3 or 5 days. WT mice had increased urine output and lowered urine osmolality after 3 and 5 days of treatment whereas PKCα KO mice had no change in urine output or concentration. Western blot analysis revealed that AQP2 expression in medullary tissues was lowered after 3 and 5 days in WT mice; however, AQP2 was unchanged in PKCα KO. Similar results were observed with UT-A1 expression. Animals were also treated with lithium for 6 weeks. Lithium-treated WT mice had 19-fold increased urine output whereas treated PKCα KO animals had a 4-fold increase in output. AQP2 and UT-A1 expression was lowered in 6 week lithium-treated WT animals whereas in treated PKCα KO mice, AQP2 was only reduced by 2-fold and UT-A1 expression was unaffected. Urinary sodium, potassium and calcium were elevated in lithium-fed WT but not in lithium-fed PKCα KO mice. Our data show that ablation of PKCα preserves AQP2 and UT-A1 protein expression and localization in lithium-induced NDI, and prevents the development of the severe polyuria associated with lithium therapy.
    Full-text · Article · Jul 2014 · PLoS ONE
  • Source
    • "Patients with bipolar disorder generally respond well to lithium treatment, but the side effects on urinary concentrating ability often cause polyuria and polydipsia. Lithium appears to enter the principal cells of the collecting duct through the epithelial sodium channel ENaC and reduces aquaporin-2 expression [29]. Long-term treatment with lithium may induce tubulo-interstitial nephritis that can lead to irreversible NDI and end-stage renal failure [30]. "
    [Show abstract] [Hide abstract] ABSTRACT: The treatment of hyponatraemia due to SIADH is not always as straightforward as it seems. Although acute treatment with hypertonic saline and chronic treatment with fluid restriction are well established, both approaches have severe limitations. These limitations are not readily overcome by addition of furosemide, demeclocycline, lithium or urea to the therapy. In theory, vasopressin-receptor antagonists would provide a more effective method to treat hyponatraemia, by virtue of their ability to selectively increase solute-free water excretion by the kidneys (aquaresis). In this review we explore the limitations of the current treatment of SIADH and describe emerging therapies for the treatment of SIADH-induced hyponatraemia.
    Full-text · Article · Nov 2009 · NDT Plus
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