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.
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.
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
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.’’
1. Freeman MP, Freeman SA (2006) Lithium: Clinical considerations in internal medicine.
Am J Med 119:478 – 481.
2. Peet M, Pratt JP (1993) Lithium: Current status in psychiatric disorders. Drugs 46:7–17.
3. Timmer RT, Sands JM (1999) Lithium intoxication. J Am Soc Nephrol 10:666 – 674.
4. Kwon T-H, et al. (2000) Altered expression of renal AQPs and Na(⫹) transporters in rats
with lithium-induced NDI. Am J Physiol 279:F552–F564.
5. Marples D, Christensen S, Christensen EI, Ottosen PD, Nielsen S (1995) Lithium-induced
downregulation of aquaporin 2 water channel expression in rat kidney medulla. J Clin
Invest 95:1838 –1845.
6. Nielsen J, et al. (2003) Segment-speciﬁc ENaC downregulation in kidney of rats with
lithium-induced NDI. Am J Physiol 285:F1198 –F1209.
7. Thomsen K, Bak M, Shirley DG (1999) Chronic lithium treatment inhibits amiloride-
sensitive sodium transport in the rat distal nephron. J Pharmacol Exp Ther 289:443– 447.
8. Kim Y.-H., et al. (2003) Altered expression of renal acid-base transporters in rats with
lithium-induced NDI. Am J Physiol 285:F1244 –F1257.
9. Christensen BM, et al. (2004) Changes in cellular composition of kidney collecting duct
cells in rats with lithium-induced. NDI Am J Physiol 286:C952–C964.
10. Christensen BM, Kim Y-H, Kwon T-H, Nielsen S (2006) Lithium treatment induces a
marked proliferation of primarily principal cells in rat kidney inner medullary collect-
ing duct. Am J Physiol 291:F39 –F48.
11. Markowitz GS, et al. (2000) Lithium nephrotoxicity: A progressive combined glomer-
ular and tubulointerstitial nephropathy. J Am Soc Nephrol 11:1439 –1448.
12. Garrido C, et al. (2006) Heat shock proteins 27 and 70: Anti-apoptotic proteins with
tumorigenic properties. Cell Cycle 5:2592–2601.
13. Doble BW, Woodgett JR (2007) Role of glycogen synthase kinase-3 in cell fate and
epithelial-mesenchymal transitions. Cells Tissues Organs 185:73– 84.
14. Manning BD, Cantley LC (2007) AKT/PKB signaling: Navigating downstream. Cell
15. Nielsen S (2002) Aquaporins in the kidney: From molecules to medicine. Physiol Rev
16. Stambolic V, Ruel L, Woodgett JR (1996) Lithium inhibits glycogen synthase kinase-3
activity and mimics wingless signalling in intact cells. Curr Biol 6:1664 –1668.
17. Klein PS, Melton DA (1996) A molecular mechanism for the effect of lithium on
development. Proc Natl Acad Sci USA 93:8455– 8459.
18. Rao R, et al. (2005) Lithium treatment inhibits renal GSK-3 activity and promotes
cyclooxygenase 2-dependent polyuria. Am J Physiol 288:F642–F649.
19. Bijur GN, De SP, Jope RS (2000) Glycogen synthase kinase-3
ine- and heat shock-induced apoptosis: Protection by lithium. J Biol Chem
20. Zelenina M, et al. (2000) Prostaglandin E(2) interaction with AVP: Effects on AQP2
phosphorylation and distribution. Am J Physiol 278:F388 –F394.
21. Hoeﬂich KP, et al. (2000) Requirement for glycogen synthase kinase-3
in cell survival
B activation. Nature 406:86 –90.
22. Novak A, Dedhar S (1999) Signaling through
-catenin and Lef/Tcf. Cell Mol Life Sci
23. Li Y, Shaw S, Kamsteeg EJ, Vandewalle A, Deen PM (2006) Development of lithium-
induced nephrogenic diabetes insipidus is dissociated from adenylyl cyclase activity.
J Am Soc Nephrol 17:1063–1072.
24. Umenishi F, Narikiyo T, Vandewalle A, Schrier RW (2006) cAMP regulates vasopressin-
induced AQP2 expression via protein kinase A-independent pathway. Biochim Biophys
Acta 1758:1100 –1105.
25. Chou CL, DiGiovanni SR, Luther A, Lolait SJ, Knepper MA (1995) Oxytocin as an
antidiuretic hormone II: Role of V2 vasopressin receptor. Am J Physiol 269:F78–F85.
26. Hoffert JD, van Balkom BW, Chou CL, Knepper MA (2004) Application of difference gel
electrophoresis to the identiﬁcation of inner medullary collecting duct proteins. Am J
Physiol 286:F170 –F179.
Nielsen et al. PNAS
March 4, 2008