Division of Nephrology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
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Osmolality of the mammalian renal medulla is high because of the operation of the urinary concentrating mechanism. To understand molecular events during the early phase of cellular adaptation to hypertonicity, we performed comprehensive searches for genes induced in response to hypertonicity using a cell line (mIMCD3) derived from the inner medullary collecting duct of mouse kidney. PCR-based subtractive hybridization of cDNA pools and cDNA microarray analysis were used. We report 12 genes whose mRNA expression is significantly increased within 4 h after exposure to hypertonicity. The increase in mRNA expression was the result of increased transcription. Many are either stress response genes or growth regulatory genes, supporting the notion that hypertonicity evokes the stress response and growth regulation in cells. Experiments using inhibitors revealed that mitogen-activated protein kinases were commonly involved in signaling for the induction of genes by hypertonicity. Tyrosine kinases and phosphatidylinositol 3-kinase also play a significant role. Signaling pathways for stimulation of transcription appeared quite diverse in that each gene was sensitive to different combinations of inhibitors.
renal medulla; p38 mitogen-activated protein kinase; complimentary deoxyribonuclease microarray analysis
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INTRODUCTION |
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OSMOLALITY OF THE mammalian kidney medulla is very high. In rat kidney medulla, the osmolality routinely exceeds 3,000 mosmol/kgH2O depending on the animal's hydration status. The high osmolality provides the driving force for water reabsorption and urinary concentration, an important function of the kidney for proper maintenance of blood pressure (25). There are two principal solutes in the interstitium of the renal medulla (NaCl and urea; see Ref. 5). Hyperosmolal NaCl and urea both induce apoptosis in a dose-dependent manner (37, 47). However, the underlying mechanisms appear different. Exposure of a cell to hyperosmolal salt (hypertonicity) results in an immediate increase in ionic strength inside the cell and double-stranded DNA breaks (29). As a result, the classical response to double-stranded DNA breaks is induced: apoptosis on one hand and activation of ATM kinase and induction and activation of p53, which in turn opposes apoptosis and arrests cell cycle progression (16), on the other hand. The balance of the two opposing pathways is determined by the degree of hypertonicity. In contrast, hyperosmolar urea does not increase cellular ionic strength nor causes double-stranded DNA breaks (29).
Cells in the renal medulla adapt to hypertonicity by accumulating
organic solutes called "compatible osmolytes" or "organic osmolytes," such as betaine, taurine, sorbitol, and
myo-inositol (21). The accumulation of
compatible osmolytes reduces the stress of hypertonicity by lowering
cellular ionic strength resulting from osmotic replacement
(5). The cellular accumulation of compatible osmolytes is
orchestrated in large part by a transcription factor named
tonicity-responsive enhancer binding protein (TonEBP, also called
NFAT5; see Ref. 39). TonEBP is stimulated by hypertonicity and, in turn, stimulates transcription of genes that encode the Na+-myo-inositol cotransporter (SMIT; see Ref.
46), the Na+-Cl-betaine
cotransporter (BGT1; see Ref. 38), and aldose reductase (AR; see Ref. 27), which are responsible for the cellular
accumulation of myo-inositol, betaine, and sorbitol,
respectively. Emerging data suggest that hypertonicity is also a signal
for tissue-specific gene expression. The vasopressin-regulated urea
transporter (UT-A) is exclusively expressed in the renal medulla and
plays a key role in accumulation of urea (4). TonEBP also
stimulates transcription of UT-A (42). Thus hypertonicity
induces a specific set of gene expression that determines the phenotype
of the renal medulla and allows cells to overcome the stress of hypertonicity.
The sensors and signaling pathways to TonEBP are not known. Because it takes several hours for induction of TonEBP in response to hypertonicity (58), there might be early genes required for this process. In addition, we are interested in uncovering the network of genes that are activated by hypertonicity. To explore these questions, we performed comprehensive searches for genes in which mRNA expression was increased immediately after exposure to hypertonicity. We report 12 genes in which transcription is stimulated within 4 h after cells are exposed to hypertonicity. Experiments with inhibitors indicate that multiple signaling pathways with a variety of protein and lipid kinases are involved in the stimulation of gene transcription.
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MATERIALS AND METHODS |
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Cell cultures and reagents. mIMCD3 cells were established from the inner medullary collecting ducts of a transgenic mouse expressing SV40 large T-antigen (43). The cells of passage number fewer than 20 were maintained on plastic tissue culture dishes in DMEM-F-12 (1:1) supplemented with 10% FBS and 2% penicillin-streptomycin. For hyperosmolality experiments, confluent cells were exposed to control isoosmolar medium or hyperosmolar medium supplemented with (in mM) 100 NaCl, 200 raffinose, or 200 urea. To study the effects of different stresses, cells were exposed for 2.5 h to heat (42°C for 1.5 h followed by a 1-h recovery period) or for 4 h to various concentrations of H2O2. For inhibitor experiments, cells were initially incubated with inhibitors in isotonic medium for 1 h and switched to isotonic or hypertonic medium (100 mM NaCl) containing the same concentration of inhibitors. Cells were further incubated for either 1 or 4 h depending on the genes studied. Each inhibitor was dissolved in DMSO, and an appropriate volume of this solution was added to the medium. The largest volume of solvent used to dissolve inhibitors was added to the medium of controls. The final concentration of DMSO in the medium was <0.1%. Actinomycin D was purchased from Sigma (St. Louis, MO). Cycloheximide, SB-203580, genistein, and LY-294002 were purchased from Calbiochem (San Diego, CA). U-0126 was purchased from Promega (Madison, WI).
SSH. mIMCD3 cells were cultured for 1 or 4 h in either isotonic or hypertonic (100 mM NaCl added) medium. Total RNA was isolated using Trizol reagent (Life Technologies, Rockville, MD), and poly(A) RNA was affinity purified on an Oligotex column (Qiagen, Valencia, CA). The poly(A) RNA were reverse transcribed, and suppressive subtractive hybridization (15) was performed to enrich cDNAs overrepresented in the hypertonic sample compared with matched isotonic sample using the PCR-Select cDNA Subtractive Hybridization kit (Clontech, Palo Alto, CA). The enriched cDNAs were recovered by PCR and cloned into pCRII TOPO vector using the T/A cloning kit (Invitrogen, Carlsbad, CA). The selected cDNA clones were screened for false positives according to the manufacturer's instructions, and the final cDNAs were further analyzed by Northern blot analysis and DNA sequencing. Nucleic and amino acid homology searches were performed using the BLAST program.
cDNA microarray analysis. With the use of a commercial service (Genome Systems, St. Louis, MO), the same pairs of poly(A) RNA used above were also analyzed to identify genes that are differentially expressed. Poly(A) RNA from isotonic and hypertonic cells were reverse transcribed separately with 5'-Cy3- and Cy5-labeled random nanomers. The labeled probes were then hybridized to a mouse GEM1 Microarray containing 8,700 cDNAs. The results were analyzed with the GEM tool 2.4 software. The cDNA clones for those genes whose expression was higher in hypertonic cells were purchased and used for Northern blot analyses.
Northern blot analysis.
An equal amount of RNA (10 µg) as determined by ultraviolet (UV)
absorbance was separated on an 1% agarose gel containing 2.2 M
formaldehyde and was transferred to a nitrocellulose membrane. Membranes were then hybridized overnight with 32P-labeled
cDNA probes. After washing for 20 min at 60°C in 0.5× saline-sodium
citrate and 0.1% SDS, radioactivity was visualized and quantified
using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). To
demonstrate equal loading of RNA, ethidium bromide staining of the blot
(shown in Figs. 1 and
2) and Northern analysis with glyceraldehyde-3-phosphate dehydrogenase probe (data not shown) were
performed with the same results.
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RESULTS |
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Identification of mRNAs upregulated in response to hypertonicity using suppressive PCR subtractive hybridization and cDNA microarray analysis. In an effort to identify genes that are stimulated early in response to hypertonicity, mIMCD3 cells were cultured in hypertonic and control isotonic medium for 1 and 4 h before poly(A) RNA was isolated. The mRNA species enriched in the hypertonic cells was identified using two different methods [suppressive PCR subtractive hybridization (SSH) and cDNA microarray analysis] as described in MATERIALS AND METHODS. SSH is an "open" system that does not require any knowledge of genes or their sequences, whereas the microarray analysis is a "closed" system that uses available gene sequences, mostly expressed sequence tags (ESTs).
From the initial enriched cDNA libraries of SSH, we obtained 150 clones from the 1-h library and 100 clones from the 4-h library. Two rounds of selections, including Northern blot analysis of RNA from isotonic and hypertonic cells, yielded 12 true positive clones from the 1-h library and 16 clones from the 4-h library. The selected cDNA clones were sequenced and BLAST matched for their identity. Some were previously reported sequences (see Table 1), whereas others were not. Of note, 12 out of the 16 4-h clones were partial cDNAs of mouse AR. Because the induction of AR by hypertonicity has been characterized extensively (21, 27), the AR clones were excluded from the subsequent experiments. Three clones out of the 12 1-h clones are partial cDNAs for amino acid transport system A2 (ATA2). Thus a total of 15 genes were identified from SSH.
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Characterization of mRNA induction by hypertonicity.
To characterize the induction of mRNA species identified above, we
performed Northern blot analysis (Fig. 1, A and
B). First, the time course of mRNA induction in response to
hypertonicity was examined (Fig. 1A). The kinetics of mRNA
induction was consistent with the data obtained by SSH and the
microarray analysis (Table 1). Expression of all the genes obtained
from the 1-h sample was induced at 1 h. Among the 1-h genes,
expression of ATA2, thrombospondin-1 (TSP-1), human liver DnaJ-like
protein (HLJ1), and inhibitory factor-B-
(I
B-
) remained
elevated at 4 h. Expression of other genes [growth-arrest and DNA
damage-inducible (GADD) 34, CYR61, and TIS11b] returned to the basal
(isotonic) level, suggesting that their function may be specific for
the acute-phase adaptive process during hypertonic stress. Expression
of the 4-h genes peaked at 4 h except tissue plasminogen activator
(t-PA) and p8.
Effects of other stresses.
Expression of many stress proteins is induced by more than one kind of
stress. For instance, expression of several heat shock proteins is
induced by hypertonicity and heat (44, 48). As shown in
Fig. 2A, seven genes were induced by heat and four genes were induced by H2O2 (i.e., oxidant stress) in
a dose-dependent manner. GADD34, IB-
, and TIS11b were induced by
both heat and H2O2, indicating that they are
general stress response genes.
Effects of actinomycin D and cycloheximide.
Because mRNA accumulation is a function of gene transcription and mRNA
stability, we asked whether transcription played a role in the
accumulation of mRNA in response to hypertonicity. In addition, the
possibility that the upregulated genes are immediate early genes was
investigated since the induction of these genes occurred within 4 h. By definition, transcription of immediate early genes is stimulated
rapidly and is not dependent on de novo protein synthesis. Confluent
mIMCD3 cells were treated with either actinomycin D (5 µg/ml) or
cycloheximide (10 µg/ml) for 1 h before being switched to
hypertonic medium for 1 or 4 h. Actinomycin D inhibited mRNA
induction of all of the 1- and 4-h genes by hypertonicity (45-95%
reduction in mRNA expression; Fig. 3,
lane 2). These data indicate that transcription rather than
increased mRNA stability is the key step for mRNA accumulation of the
upregulated genes in response to hypertonicity.
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Effects of kinase inhibitors.
Exposure of cells to hypertonicity leads to immediate and vigorous
activation of a number of kinases, including the three major families
of mitogen-activated protein kinase (MAPK), tyrosine kinases, and
phosphatidylinositol 3-kinase (PI3K; see Refs. 6, 28, 54, 61). We used inhibitors
to investigate the role of the kinases in the same way we used
actinomycin D and cycloheximide (see above). A given inhibitor was
scored as inhibitory (see Fig. 5, bottom) only if it
exhibited a statistically significant inhibition on both absolute
expression of mRNA in the hypertonic condition (Fig.
4) and a degree of induction of mRNA
expression (Fig. 5).
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DISCUSSION |
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Using SSH and cDNA microarray analysis, we identified 34 genes in which transcripts were upregulated early when cells are exposed to hypertonic medium. Only those genes (Table 1) in which function has been described, except AR, were selected and characterized. Many of them are either general stress response genes or growth regulatory genes, in line with other reports that hypertonicity results in the stress response and perturbation of cell growth (16, 29, 37, 47). It is likely that the increase in mRNA abundance results in a corresponding increase in protein abundance. In case of TonEBP, a threefold increase in mRNA abundance in response to hypertonicity leads to a threefold increase in synthesis of TonEBP (58).
Eight of the selected genes, including all of those induced at 1 h
after exposure to hypertonicity, are immediate early genes in that
their induction by hypertonicity was not prevented by cycloheximide, an
inhibitor of protein synthesis. Interestingly, many of them (GADD34,
CYR61, TIS11b, HLJ1, IB-
, and FHL2) were induced by cycloheximide
in isotonic conditions (Fig. 3). Induction of these genes by
hypertonicity might be caused by inhibition of protein synthesis per se
because hypertonicity is known to inhibit the overall rate of protein
synthesis (11).
Induction of the ATA2 gene by hypertonicity. Three cDNA clones identified from SSH were the mouse ortholog of human (22) and rat (53, 60) ATA2 (or SAT2). ATA2 is ubiquitously expressed in mammalian tissues, including kidney and brain. It mediates Na+-coupled cellular uptake of short-chain neutral amino acids such as alanine, serine, proline, and glutamine. Its induction in response to hypertonicity and its putative role as a compatible osmolyte transporter have been described in the literature (10, 12, 59). The induction and shut off of ATA2 are much faster (Fig. 1A) than those of SMIT, BGT, and AR (21, 58). This observation is consistent with the hypothesis (12) that the "regulatory volume increase" that occurs immediately after exposure to hypertonicity is mediated by the accumulation of neutral amino acids. Subsequently, neutral amino acids are replaced by inositol, betaine, and sorbitol as the activity of SMIT, BGT1, and AR increases. In addition, its robust expression in brain (22, 53, 60) raises the possibility that ATA2 contributes to the maintenance of brain cell volume during hypernatremia.
Induction of stress response and growth regulatory genes by
hypertonicity.
During the early phase of exposure to hypertonicity, the structure and
function of proteins are perturbed (44). It has been postulated that heat shock proteins protect the cell by stabilizing protein conformation until compatible osmolytes accumulate to sufficient levels (44, 48). Hypertonicity induces the
expression of many heat shock proteins, including Hsp70,
B-crystallin, Hsp110, and Osp94 (44, 48). There
is a renal corticomedullary gradient of Hsp70, Hsp25, and Osp94
expression that parallels the osmotic gradient (40).
In this regard, our discovery of the early induction of HLJ1 by
hypertonicity is highly significant. HLJ1 is a member of the Hsp40
family and is expressed ubiquitously, including in the kidney
(23). The members of the Hsp40 family function in association with Hsp70 molecular chaperones to facilitate protein folding (26). As cochaperones, they recruit Hsp70
partners and augment their binding to their protein
substrates by accelerating the ATP hydrolysis step of the chaperone
cycle. The simultaneous induction of Hsp40-Hsp70 functional
partners reinforces the notion that hypertonicity is a form of stress
that denatures proteins.
Induction of the genes involved in anticoagulation and fibrinolysis by hypertonicity. Heparan sulfate D-glucosaminyl 3-OST-1 converts nonanticoagulant heparan sulfate to anticoagulant heparan sulfate by transferring a sulfate group to the 3-OH position of glucosamine residues (34). The anticoagulant heparan sulfate binds to and activates antithrombin, which is an important natural anticoagulant. t-PA is a serine protease that converts the blood zymogen plasminogen to more active plasmin and degrades the fibrin clot. In HeLa and human umbilical cord endothelial cells, t-PA mRNA expression is stimulated by hypertonicity (33), as in mIMCD3 cells reported here. It is possible that 3-OST-1 and t-PA are TonEBP target genes like SMIT and BGT1 because their induction is slow (Fig. 1A) and sensitive to cycloheximide.
Other genes induced by hypertonicity. A member of the Eph family of receptor tyrosine kinases, the EphR A2, is widely expressed in the central and peripheral nervous system (17). The ephrin receptors and their ligands play a role in axon guidance, cell migration, and boundary formation between groups of cells during development of the nervous system. FHL2 is a member of the FHL protein family (9). The hallmark of the FHL proteins is the presence of four LIM domains and a LIM half-motif located at the amino terminus of the protein. The LIM domain is a cysteine-rich, double-zinc motif involved in protein-protein interaction (2) and regulation of transcription (13). The physiological implication of the induction of EphR A2 and FHL2 by hypertonicity is not known. Because both are also induced by heat shock (Fig. 2), they may be general stress response genes.
Involvement of multiple signaling pathways in gene induction by hypertonicity. In 11 of the 12 genes studied, gene induction by hypertonicity was inhibited by SB-203580 (Figs. 4 and 5). This is significant in that p38 MAPK is implicated in adaptation to hypertonicity from yeast to mammals. The yeast ortholog of p38 MAPK, named Hog1p, plays a key role in adaptation to hypertonicity (8). Hog1p is part of the protein kinase cascade (35) that relays the two osmosensors in the plasma membrane to the early phase of GPD1 stimulation (45). Gpd1p is a key enzyme in biosynthesis of glycerol, the major compatible osmolyte of yeast (8). In mammalian cells, SB-203580 inhibits TonE-mediated induction of BGT1, SMIT, and HSP70 (14, 41, 49), indicating that p38 MAPK is involved in signaling to TonEBP. Therefore, the p38 MAPK-sensitive genes reported here are candidate early genes in the signaling pathways to TonEBP.
In contrast to SB-203580, other inhibitors inhibited the gene induction only in distinct subsets of genes (Figs. 4 and 5). With the exception of GADD34 and TIS11b, a different combination of inhibitors was effective for each gene, indicating a unique signaling mechanism. It should be noted that SB-203580 inhibits other protein kinases and signaling molecules in addition to p38 MAPK (7, 31). Thus the genes that are sensitive to SB-203580 do not necessarily share the same signaling pathways. Related to this, hypertonicity causes double-stranded breaks of DNA and, as a result, activates a number of DNA damage-dependent protein kinases such as ATM and DNA-dependent protein kinase (16, 29). Because LY-294002 and wortmannin also inhibit these kinases (51), their role should be considered for the effects of LY-294002 in addition to that of PI3K. Collectively, these data indicate that signaling pathways for stimulation of gene transcription in response to hypertonicity are quite diverse, involving a variety of protein and lipid kinases. ![]() |
ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-42479 (to H. M. Kwon), National Research Service Award DK-9960 (to O. Nahm), and a fellowship from the Juvenile Diabetes Foundation International (3-1999-727; to S. K. Woo).
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FOOTNOTES |
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* O. Nahm and S. K. Woo contributed equally to this work.
Address for reprint requests and other correspondence: H. M. Kwon, 963 Ross Bldg., 720 Rutland Ave., Baltirmore, MD 21205 (E-mail: mkwon{at}jhmi.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published September 21, 2001; 10.1152/ajpcell.00267.2001
Received 15 June 2001; accepted in final form 28 August 2001.
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