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Cellular Response to Hyperosmotic Stresses

National Heart, Lung, and Blood Institute, Bethesda, Maryland

ABSTRACT

I. INTRODUCTION

II. PERTURBING EFFECTS OF HYPEROSMOLALITY

A. Cell Cycle Arrest

B. Apoptosis

C. DNA Damage

D. Oxidative Stress

E. Inhibition of Transcription and Translation

F. Mitochondrial Depolarization

III. HIGH NaCl: HYPERTONICITY

A. Many Cellular Components and Functions Are Affected by Hypertonicity

B. Cytoskeletal Rearrangement in Response to Hypertonicity

IV. ORGANIC OSMOLYTES ACCUMULATE IN RESPONSE TO HYPERTONICITY

A. Sorbitol

B. Betaine

C. Inositol

D. Taurine

E. GPC

V. ACTIVATION OF THE TRANSCRIPTION FACTOR TonEBP/OREBP BY HYPERTONICITY

A. TonEBP/OREBP mRNA and Protein Abundance

B. Dimerization of TonEBP/OREBP

C. Phosphorylation of TonEBP/OREBP

D. TonEBP/OREBP Nuclear Localization

E. TonEBP/OREBP Transactivational Activity

F. Interactions Between Proteins That Regulate TonEBP/OREBP

G. Inhibition of TonEBP/OREBP

H. DNA Breaks, ROS, and Cytoskeletal Perturbations as Regulators of TonEBP/OREBP

VI. ADAPTATION TO CHRONIC HYPERTONICITY

VII. SENSORS AND SIGNALERS OF HYPERTONICITY

VIII. HIGH UREA

A. Cellular Components and Functions Affected by High Urea

B. Counteraction of Urea Effects by GPC

C. Mutually Protective Effects of High Urea and High NaCl

IX. WHY IS OSMOTIC TOLERANCE OF RENAL MEDULLARY CELLS GREATER IN VIVO THAN IN CULTURE?

X. OSMOPROTECTION BY GENERAL STRESS PROTEINS IN CELL CULTURE AND IN VIVO

A. Gadd45

B. p53

C. Heat Shock Proteins

D. Heme oxygenase 1

XI. SUMMARY AND CONCLUSIONS

GRANTS

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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Our interest in hyperosmolality rose from consideration of the extraordinary osmotic stress that cells experience in the renal medulla. There, they are exposed to much higher interstitial levels of NaCl and urea than are cells anywhere else in the body, the high concentrations being associated with the role of the renal medulla in the urinary concentrating mechanism. Interstitial composition is estimated from that of plasma, since the two are closely related. At the tip of the renal inner medulla of antidiuretic male hamsters, representative concentrations in interstitial fluid (measured by micropuncture of vasa recta) are 697 meq/l sodium, 614 mM urea, and 1,744 mosmol/kgH₂O osmolality (189). In contrast, normal levels in peripheral plasma are much lower, namely, 128 meq/l sodium and 8 mM urea (199). High NaCl and urea are stressful to cells, altering their function or even killing them by apoptosis. In cell culture (198, 257), the apoptosis occurs at levels much lower than those that normally exist in the renal medulla. So, how do cells in the renal medulla survive? The answer, it turns out, is complex and becoming increasingly so. Many of the studies of osmotic stress have been with renal cells, especially renal medullary cells. However, the effects of osmotic stress have also been examined with many other mammalian cell types (Table 1), and numerous different changes have been observed in them. Some of the changes have been reported in a few or only one cell type. These could depend on the initial differentiated phenotype of the particular cells. However, some changes have been observed in a variety of cells, pointing to some general principles that may apply to all cells. The fact that cells not normally exposed to high NaCl and urea share these general responses suggests that hyperosmolality may be such a basic threat that all cells are prepared to confront it, whether they will ever experience it or not. In this review we concentrate mainly on renal epithelial cells, recognizing that osmotic stress has also been studied in many other cell types (Table 1).

View larger version (24K): [in this window] [in a new window]   FIG. 1. Cellular adaptation to hypertonic stress.

	II. PERTURBING EFFECTS OF HYPEROSMOLALITY

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Acute elevation of NaCl or urea produces rapid arrest in all phases of the cell cycle. The cells in G₁ do not proceed to initiate DNA replication. Those in S stop replicating their DNA. Those in G₂ do not divide. The duration of the arrest depends on how high the NaCl or urea is. The arrest is shortest in G₁ and S, so cells accumulate in G₂ (198). By the time that the arrest ends, the cells are sufficiently adapted so that can they proliferate through many passages at the elevated osmolality. The rate of proliferation of adapted cells is directly related to the osmolality (198). When NaCl is elevated to 400–500 mosmol/kgH₂O, proliferation is the same as at 300 mosmol/kgH₂O, but at higher levels it decreases progressively. Finally, there is a limit as to how high a concentration the cells can withstand. Beyond this they die, as discussed in section IIB. Cell cycle arrest provides time for cells to adapt to higher osmolality by accumulation of compatible and counteracting organic osmolytes (see sects. IV and VIIIB) and increased expression of heat shock proteins (see sect. X). In the absence of such protective adaptation, the cells die.

DNA damage is a well-known cause of cell cycle arrest. High NaCl increases the number of DNA breaks in cells (see sect. IIC), and some of the same signals that arrest the cell cycle in response to DNA damage from other causes also play a role in high NaCl-induced cell cycle arrest. Delay in G₁ and S depends on activation of p53 following both high NaCl (72) and ionizing radiation (IR) (100). In the case of high NaCl, the delay in S is sensitive to caffeine, implying involvement of ATM/ATR kinases (72), which are targets of p53 (100). Delay in G₂ depends on activation of p38 following both high NaCl (74) and ultraviolet (UV) radiation (28).

Despite these similarities, there are major differences between the cell cycle delay caused by high NaCl and that from other causes. DNA breaks from the other causes are repaired during the cell cycle arrest, but those caused by high NaCl are not (see sect. IIC). Furthermore, the mechanisms differ. Phosphorylation of Chk1 is important for the cell cycle arrest induced by the DNA damage from other stresses (173), but high NaCl does not cause phosphorylation of Chk1 and, in fact, prevents its phosphorylation in response to DNA damage caused by UV or IR (73). Also, inhibition of Chk1 by UCN-01 prevents the cell cycle delay caused by other DNA damaging agents, but not the cell cycle delay caused by high NaCl (73). The question remains as to why the increased DNA breaks that persist in cells adapted to high NaCl do not activate cell cycle checkpoints. Possibly, they are modified so that they are invisible to the DNA damage sensors or the DNA damage response is inhibited.

The cell cycle arrest induced by high urea closely resembles that induced by high NaCl (198), but less is understood about its mechanism. Urea does not activate some of the DNA damage response proteins involved in the cell cycle arrest induced by high NaCl, such as p53 (72) and p38 (149, 251). High urea increases phosphorylation of ATM in HEK293 cells (124), but we do not know whether this is involved in the accompanying cell cycle arrest.

B. Apoptosis

Cell death results when acute hypertonicity exceeds a certain level. The level differs between cell lines, but is rather sharply defined in each. Above that level, massive cell death occurs within several hours. The dying cells exhibit classical features of apoptosis (25, 94, 198, 257). DNA condenses and apoptotic bodies appear, the DNA becomes fragmented with appearance of a "DNA ladder" in gel electrophoresis, the broken DNA ends are labeled in TUNEL assays, and phosphatidylserine becomes exposed at the cell surface. There are two basic pathways for apoptosis: an intrinsic (mitochondrial) pathway and an extrinsic (death receptor) pathway (132). There is evidence favoring both of these pathways as a cause of hypertonicity-dependent apoptosis. In favor of the intrinsic pathway is that hypertonicity causes a rapid mitochondrial dysfunction (see sect. IIF) that precedes apoptosis. Thus acutely exposing mIMCD3 cells to a lethal level of osmolality (700 mosmol/kgH₂O, NaCl added) rapidly depolarizes their mitochondria, decreases mitochondrial Bcl2/Bax ratio, and increases cell ADP/ATP ratio (197). However, the mitochondria do not release cytochrome c, and although their Bcl2/Bax ratio decreases, it also decreases at 500 mosmol/kgH₂O without any subsequent apoptosis. In favor of the extrinsic pathway is that acute exposure of HeLa cells to 900 mosmol/kgH₂O (sorbitol added) causes clustering and internalization of tumor necrosis factor- (TNF-) receptors (253). Also, increasing osmolality to 500 mosmol/kgH₂O by adding NaCl causes apoptosis in macrophages (U937) and B lymphocytes (LCL721), accompanied by increased production of TNF-, suggesting that they produce their own death ligand and self-destruct (162). Neutralization of the TNF- by a specific antibody protects the U937 cells from apoptosis, but it does not protect the LCL721 cells, indicating that the autogenous TNF- could be triggering the apoptosis in the former, but not in the latter. In another example, increasing the osmolality bathing primary rat hepatocytes to 405 mosmol/kgH₂O (NaCl added) causes trafficking of CD95 to the plasma membrane, accompanied by caspase-8 and -3 activation; however, this only amounts to a priming for apoptosis because full-blown apoptosis (DNA fragmentation and phosphatidylserine redistribution) does not occur unless exogenous CD95 ligand is also added (244). We conclude that, since hypertonicity has been observed to trigger components of both the intrinsic and extrinsic pathway to apoptosis in one type of cell or another, it is premature to conclude that either pathway is always involved.

The fact that both hypertonicity and apoptosis cause cells to shrink has led to recognition that the processes are linked. Thus apoptotic volume decrease (AVD) is an early event in apoptosis. It precedes cytochrome c release from mitochondria, and it is maintained until the cell fragments (184, 224). Inhibition of AVD prevents apoptosis from going to completion (184, 224). Regulatory volume increase (RVI) is the usual initial response to cell shrinkage by hypertonicity (311). During RVI, electrolytes and water are taken up into the cell, restoring its volume. An ion carrier (NKCC1) and ion exchangers (NHEs) are activated in RVI (Table 1 and Ref. 311). Although RVI occurs in cells shrunken by hypertonicity, it is impaired in cells undergoing an AVD initiated either by a death receptor ligand (Fas) or an by an inducer of mitochondria-mediated apoptosis (staurosporine) (185). Given that AVD is an early event in apoptosis and that RVI does not occur, it seemed possible that cell shrinkage induced by hypertonicity, if uncompensated by RVI, might by itself cause apoptosis (24, 25). This idea is supported by a number of observations. RVI is impaired in T lymphocytes, and they undergo apoptosis at a much lower level of tonicity than do cells proficient in RVI (25). CHO (PC120) cells, which are deficient in RVI because they lack NHE-1, are particularly susceptible to hypertonicity-induced apoptosis. Reconstituting them with NHE-1 restores RVI and protects them (185). Finally, inhibition of NHE by amiloride sensitizes HeLa (185) and renal tubular epithelial (RTC) cells (319) to hypertonicity-induced apoptosis. Taken together, these observations indicate that hypertonicity-induced shrinkage, if uncompensated by RVI, can cause apoptosis. What is lacking at this point is an understanding of how cell shrinkage activates the executioners of apoptosis. Although hypertonicity shrinks cells immediately, several hours elapse before the final stages of apoptosis are reached (25, 88, 185, 197). Interestingly, when cells are exposed to levels of hypertonicity that would be deadly if prolonged, returning the cells to normotonicity after a brief time (0.5–1 h) prevents apoptosis (24; Dmitrieva NI and Burg MB, unpublished results). Thus there must some additional processes between the initiation of hypertonicity and the point of no return from apoptosis. Those processes apparently include activation of Rac and p38, followed by phosphorylation and nuclear translocation of p53 (88).

C. DNA Damage

Acute elevation of osmolality from 300 to 500–600 mosmol/kgH₂O by adding NaCl to cells causes DNA strand breaks in mIMCD3 cells (73, 153), accompanied by cell cycle arrest in all phases of the cell cycle (see sect. IIA). The cell cycle arrest that is associated with most other causes of DNA damage provides a lull in DNA replication during which the DNA can be repaired (345). Thus it seemed logical that, during the transient cell cycle arrest that follows acute elevation of NaCl, the DNA breaks would be repaired. Surprisingly, they are not. Numerous DNA breaks persist in cells that have adapted to high NaCl and reentered the cell cycle (75). Despite these DNA breaks, the adapted cells proliferate rapidly, appear normal, and exhibit little, if any, apoptosis (42, 75, 259). Even more surprising, there are numerous DNA breaks in cells in the mouse renal inner medulla as long as its interstitial NaCl concentration remains at its normal high level (75).

High urea causes a different sort of DNA damage. Neither acute (153, 153) nor chronic (75) exposure of cells to urea causes DNA strand breaks. Furthermore, addition of urea does not significantly increase the number of breaks caused by high NaCl (75), implying that NaCl, alone, is responsible for the DNA breaks in the normal renal inner medulla. However, high urea does produce oxidative DNA damage, namely, 8-oxoguanine lesions (337). If 8-oxoguanine lesions are not repaired, they produce mutations during DNA replication (104). Thus renal medullary cells have DNA breaks caused by high NaCl and oxidative lesions caused by high urea. Nevertheless, the cells survive and function in vivo and, following adaptation, proliferate rapidly in culture.

Normally, DNA breaks attract and activate a protein complex that rapidly repairs the DNA. If the repair fails, the cells die by apoptosis. Cells deficient in components of the DNA repair system are very sensitive to extrinsically caused DNA damage and may not be viable because they cannot repair DNA breaks that occur during normal replication or transcription (173, 183, 320, 349). Remarkably, DNA breaks induced by high NaCl persist as long as NaCl remains high. Yet, despite the DNA breaks, apoptosis is not evident, and the cells proliferate rapidly in culture (75). Reducing NaCl back to normotonic levels in culture (73) and in vivo (75) results in rapid repair of the breaks, implying that high NaCl inhibits DNA repair. Direct proof for this came from measuring the rate of repair of luciferase reporter plasmids damaged by UV before they were transfected into cells. The repair is much faster at 300 mosmol/kgH₂O than when NaCl is added to 500 mosmol/kgH₂O (73, 75).

Although it is clear that hypertonicity induces DNA damage, the molecular basis is poorly understood. Hypertonicity increases expression or activity of several DNA damage response proteins including Gadd45 (see sect. XA), p53 (see sect. XB), ATM (see sect. V, D and E), and chk2 (264). Other DNA damage response proteins are not affected. Thus other genotoxic agents that induce DNA breaks cause phosphorylation of Chk1, signaling cell cycle arrest (27, 122), but high NaCl does not (73, 75, 264). The effect of high NaCl on some of the other DNA damage response proteins is controversial. Thus Mre11 exonuclease is a nuclear protein that normally binds rapidly to DNA breaks, beginning the process of repair (64), and histone H2AX becomes phosphorylated at DNA double-stranded breaks caused by a number of agents (90, 250, 309), both being prerequisite for successful DNA repair (45, 229). In some studies, high NaCl decreased the chromatin-bound fraction of Mre11 (76) and caused it to move from the nucleus to the cytoplasm, where it is unavailable to initiate DNA repair (73, 75). Also, phosphorylation of histone H2AX was not induced (73, 75). These aspects of the DNA damage response became activated immediately when NaCl was reduced. When osmolality was lowered from 500 to 300 mosmol/kgH₂O, Mre11 moved back into the nucleus, histone H2AX and Chk1 became phosphorylated, and the DNA breaks disappeared within a few hours. These results are consistent with the idea that high NaCl inhibits those particular components of DNA damage response or that the response to high NaCl modifies or camouflages DNA breaks so that they are not recognized by the usual DNA damage response system. However, in another study, Mre11 stayed in the nucleus after NaCl was increased and histone H2AX became phosphorylated (264). Why the results differ remains unresolved. The authors of the latter study (264) speculated that the transient H2AX phosphorylation that they observed indicated repair of any DNA double-strand breaks, but they did not actually measure the nature or extent of DNA damage. In summary, direct measurements demonstrate that high NaCl increases the number of DNA breaks both in cell culture and in vivo. However, the exact nature of the breaks, the mechanism by which they are induced, and the details of the cellular response remain to be settled.

The DNA-protein kinase (DNA-PK) complex, which contains Ku86, Ku70, and DNA-PKcs, contributes to repair of DNA double-strand breaks by nonhomologous end joining (NHEJ) (193, 222, 274). When cells that lack Ku86 are exposed to high NaCl, they have aberrant mitosis, slower growth, and increased chromatin fragmentation compared with wild type cells (76).

D. Oxidative Stress

Elevation of NaCl or urea causes oxidative stress (328, 337, 340, 347, 348, 352) with elevation of ROS (328, 337, 348). ROS can modify DNA bases by forming 8-oxoguanine (104) and can carbonylate proteins (170). Mutations result when DNA containing 8-oxoguanine lesions is replicated (104). Carbonylation affects protein stability and function, notably inhibiting enzyme activity (279). In cell culture, carbonylation occurs within 5 min and with addition of as little as 5 mM urea, which is the normal level in plasma. Since carbonylation increases as urea is raised over the range found in uremia (337), urea apparently can contribute directly to the carbonylation found in that condition. Strikingly, a high level of protein carbonylation is present in vivo in inner medullary cells (337), which are normally exposed to high NaCl and urea. Carbonylated proteins are not repaired. Instead, they undergo accelerated degradation by proteasomes and are replaced (169). At this point we do not know what the functional consequences are of urea- and NaCl-induced oxidation of renal medullary proteins, but it is of obvious interest to find out. Oxygen tension is low in the renal inner medulla because its countercurrent exchange system limits the access of oxygen. Glycolysis predominates there (9). The low oxygen tension and respiration may minimize the oxidative stress in this tissue.

ROS can be destructive, but they also have important signaling functions (reviewed in Ref. 87). Several observations support a role for ROS in signaling osmoprotective responses: 1) antioxidants that reduce superoxide suppress high NaCl-induced increases of both TonEBP/OREBP transcriptional activity and expression of BGT1 mRNA (see sect. VH)(347, 348). 2) The antioxidant N-acetyl-L-cysteine (NAC) prevents urea-induced increase of mRNA and protein abundance of the oxidative stress-responsive transcription factor Gadd153/CHOP (340). 3) NAC also prevents both high NaCl-induced phosphorylation of ERK1/2 and p38 and increase of COX-2 protein expression (328). 4) Lowering ROS production by mitochondrial inhibitors reduces high NaCl-induced increase of COX-2 (328).

E. Inhibition of Transcription and Translation

High NaCl reversibly inhibits synthesis of DNA, RNA, and protein in cultured cells (248). Synthesis recovers within 10 min if NaCl is lowered (248) and more slowly as cells adapt to sustained hypertonicity (235). The effect on DNA synthesis has already been discussed in section IIA. That on transcription and translation is discussed in what follows.

Hypertonicity rapidly and reversibly inhibits RNA synthesis in HeLa cells (248). Increasing osmolality to 450 mosmol/kgH₂O by adding NaCl inhibits synthesis of heterogeneous nuclear RNA (mRNA precursor), whereas higher concentrations of NaCl are necessary to inhibit synthesis of ribosomal and transfer RNA (232).

Hypertonicity also profoundly inhibits protein synthesis (248, 254). The inhibition is independent of the solute added (NaCl, KCl, or sucrose), occurs within several minutes, and reverses completely when normotonicity is restored. Inhibition was observed in HeLa cells (254), in chicken embryo fibroblasts (235), and in Madin-Darby canine kidney (MDCK) cells (58). Translation of globin mRNA by rabbit reticulocyte lysate in vitro is inhibited when osmolality is increased by adding NaCl, KCl, CH₃CO₂Na, or CH₃CO₂K, but not by adding the compatible osmolytes betaine or myo-inositol (26). Taken together, these results suggest that hypertonicity-induced increase of intracellular inorganic ions inhibits translation, but the inhibition abates as the inorganic ions are replaced by compatible organic osmolytes (see sect. IV) (26). Inhibition of translation could be responsible for the cell cycle arrest. The onset and ending of the cell cycle arrest (72) coincide temporally with the changes in protein synthesis (248, 254). A possible link is that cyclins are translationally regulated.

Hypertonicity reduces translation by inhibiting polypeptide chain initiation (248, 254, 313), whereas elongation and termination of those polypeptides already initiated generally proceed normally (254). Although hypertonicity inhibits translation of most cellular proteins, translation of some, such as heat-shock proteins, actually increases (see sect. XC) (209). The mechanism by which hypertonicity inhibits translation is not well understood, but there are some suggestive findings. Initiation factor eIF2 contributes to inhibition of translation by hypertonicity in reticulocytes and fetal liver nucleated erythroid progenitors (181). In those cells hypertonicity causes autophosphorylation and activation of heme-regulated inhibitor (HRI), which in turn phosphorylates and activates eIF2. The activated eIF2 inhibits translation (181). Other unidentified eIF2 kinases may also contribute (181). The lengths of untranslated regions (UTRs) in mRNAs and their secondary structure may also play a role. With the use of the rat preproinsulin II-coding sequence linked to the 5'-untranslated sequence of the message that encodes viral SV40, insertion of a hairpin barely affects translation in normotonic medium, but adding NaCl to increase osmolality by 240–300 mosmol/kgH₂O virtually abolishes translation (148). When a 136-nucleotide unstructured sequence is placed just upstream from the hairpin, translation in hypertonic medium is restored. Thus leader length and secondary structure can have opposing effects on translational efficiency under hypertonic conditions (148).

F. Mitochondrial Depolarization

Increasing osmolality by adding NaCl perturbs mitochondria in mIMCD3 (197) and Vero (60) cells, although the effects differ in detail. In the mIMCD3 cells, increasing osmolality to 700 mosmol/kgH₂O causes mitochondrial depolarization, but increasing it to only 500 mosmol/kgH₂O does not. Nuclei become condensed and caspase-9 activity increases at 700 mosmol, indicative of apoptosis, but this does not occur at 500 mosmol/kgH₂O. Electron microscopy does not reveal any mitochondrial structural changes at either of the elevated osmolalities. Although NADH exits the mitochondria at 700 (but not at 500) mosmol/kgH₂O, cytochrome c remains in the mitochondria at all osmolalities. In the Vero cells, high NaCl causes rapid mitochondrial depolarization, as in mIMCD3, but the effect occurs at a much lower osmolality. Increasing the osmolality by as little as 60 mosmol/kgH₂O depolarizes mitochondria in the Vero cells, whereas increasing it by 200 mosmol/kgH₂O does not in mIMCD3 cells. At 600 mosmol/kgH₂O mitochondria rapidly fragment in Vero cells, but the effect reverses when NaCl is lowered. In contrast, the mitochondria remain intact in mIMCD3 cells. High NaCl does not cause cytochrome c to exit from the mitochondria in either cell type. Given these results, it is not clear whether mitochondrial depolarization is a rapid response of viable cells to hypertonicity (Vero cells), or whether it is an early event in apoptosis (mIMCD3 cells). Possible mechanisms for the depolarization are reviewed in Reference 197. We conclude that high NaCl greatly perturbs mitochondria, but the significance of this effect and its mechanism remain uncertain.

	III. HIGH NaCl: HYPERTONICITY

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By definition, hypertonicity occurs when an added solute causes osmotic efflux of water from a cell and, thus, reduces cell volume. This requires a gradient of activity of the added solute across the cell membrane and semipermeability of the membrane. In accord with this definition, many experiments that test the effects of hypertonicity consist of acutely adding an impermeant solute, then observing the result over a period of minutes to hours. Since most cells have a high water permeability, water generally effluxes rapidly, and the osmotic shrinkage elevates the concentration of all intracellular components. As a result, macromolecular crowding increases, which greatly elevates the activity of cellular macromolecules (96). Also, intracellular ionic strength increases, which also affects most biochemical processes. Finally, the decreased volume is accompanied by alterations in the cytoskeleton. These are primary effects of hypertonicity, from which we presume other effects follow, although specifying the connections remains a challenge.

High NaCl is hypertonic, and NaCl is the principal determinant of tonicity of the extracellular fluid. Although cell membranes are permeable to Na, the Na⁺-K⁺-ATPase transports Na out of cells, which maintains the normal cell volume. In examining the effects of high NaCl, however, we also have to keep in mind that in addition to raising tonicity, high Na and Cl may also have specific effects. For example, elevated chloride specifically increases the expression of -Na⁺-K⁺-ATPase (43). Thus, although cellular changes caused by high NaCl may generally be due to hypertonicity, this is not necessarily so. For that reason, poorly permeating organic solutes like sorbitol, mannitol, or raffinose are often used to produce hypertonicity (Table 1). Of course, these also may have specific effects. Therefore, to establish definitively that a particular effect is caused by hypertonicity, hyperosmolality should be tested adding more than one different solute.

A. Many Cellular Components and Functions Are Affected by Hypertonicity

Table 1 lists 200 cellular components that change with osmolality, including mRNAs and proteins identified by Northern analysis, ribonuclease protection, RT-PCR, or Western analysis. The number of constituents that are affected by osmolality is too great to consider them all individually in detail. Also, caution should be exercised in interpreting the findings of those studies in which osmolality is increased by 300 mosmol/kgH₂O or more. That degree of hyperosmolality can lead to apoptosis (198, 257), so viability of cells is questionable under those circumstances. The different categories affected by tonicity (Table 1) include ion and water channels, transporters, heat shock proteins, other stress proteins, cellular and extracellular structural proteins, transcription factors and coactivators, kinases, phosphatases, other signaling proteins, enzymes, hormones, receptors, growth-related proteins, second messengers, and organic osmolytes. Determining the significance of any particular change requires additional information since it could represent an osmoprotective response, but it could also simply represent a dysfunction caused by hypertonicity. Many of these changes were discovered when an investigator added hypertonicity to the list of different stresses being applied to a component that initially was of interest for reasons unrelated to tonicity.

Screening methods have also been used to identify mRNAs and proteins affected by hypertonicity. The effect of hypertonicity on the abundance of cellular mRNAs in mIMCD3 cells was examined by using microarrays (294). When 200 mM mannitol or 100 mM NaCl is added for 6 h, 12% of 12,000 transcripts are downregulated by mannitol and NaCl, and 4% of transcripts are upregulated. As might be expected, the genes identified in this manner generally agree with those identified by other techniques (Table 1). The effect of hypertonicity on cellular proteins was examined by proteomic techniques. In one study (305) mIMCD3 cells were examined by two-dimensional gel electrophoresis (2D-GE) combined with MALDI-TOF/TOF mass spectrometry. Raising osmolality by 300 mosmol/kgH₂O (NaCl added) increases abundance of some proteins and decreases others. Several molecular chaperones are induced, such as _B-crystallin, two Hsp70 isoforms, and the osmotic stress protein (Osp94). Also, some isoforms of the translation elongation factors eEF2 and eEF1A1 are induced. With TLAH cells (70), osmolality was increased by 300 mosmol/kgH₂O for 35 h by adding NaCl. Proteins were examined by 2D-GE and MALDI-MS to determine changes in abundance; 25 proteins increase and 15 proteins decrease. Besides aldose reductase, many other metabolic enzymes like glutathione S-transferase, malate dehydrogenase, lactate dehydrogenase, -enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase increase. The cytoskeleton proteins and cytoskeleton-associated proteins vimentin, cytokeratin, tropomyosin 4, and annexins I, II, and V also increase, whereas tubulin and tropomyosins 1, 2, and 3 decrease. Vimentin, cytokeratin, and tropomyosin remained elevated in TAHL-NaCl cell lines adapted to high NaCl, as determined by Western analysis. The heat shock proteins -crystallin chain B, HSP70, and HSP90 and endoplasmic reticulum stress proteins like glucose-regulated proteins (GRP78, GRP94, and GRP96), calreticulin, and protein-disulfide isomerase decrease.

Since we do not have space to discuss all of these changes in detail, we will restrict ourselves to some that are known to be involved in osmoregulation and osmoprotection, including cellular structural proteins (see sect. IIIB), organic osmolytes (see sect. IV), stress proteins (see sect. X), the transcription factor TonEBP/OREBP (see sect. V), and the molecules that signal its activation (see sect. VII).

B. Cytoskeletal Rearrangement in Response to Hypertonicity

Hypertonicity induces rapid F-actin polymerization and remodeling of the actin cytoskeleton (35, 68, 322). However, the pattern of remodeling varies between cell types. In native rat MTAL cells (35) and glial cells (207), hypertonicity induces redistribution of F-actin from the cortical ring to a diffuse network of actin bundles, whereas in fibroblasts and HL60 cells the cortical actin ring becomes denser (68, 110). The assembly is induced by the combined action of small GTPases (Rac and Cdc42), an actin binding protein (cortactin), and a regulator of F-actin nucleation (the Arp2/3 complex), all of which form a complex with actin (68). Hypertonicity also causes rapid movement of myosin IIB to the cortical region, just beneath the plasma membrane, dependent in part on Rho kinase and p38 (230).

Integrins are a large family of heterodimeric transmembrane glycoproteins that attach cells to extracellular matrix proteins of the basement membrane. Integrin ₁₁ is a major collagen-binding receptor that is highly expressed in renal tubular cells. Hypertonicity increases integrin ₁ mRNA and protein in MDCK cells (269). Increased expression of this integrin is involved in hypertonicity-induced activation of the osmoprotective transcription factor TonEBP/OREBP (see sect. VH). Furthermore, inactivation of integrin-substratum interactions by using the integrin-binding peptide GRGDTP inhibits the hypertonicity-induced increase in glutamine transport that is mediated by phosphatidylinositol 3-kinase (PI3K) in skeletal muscle (180).

Phosphatidylinositol phosphate 5-kinase (PIP5K) kinase (322) contributes to hypertonicity-induced cytoskeletal rearrangement. Its lipid kinase activity catalyzes phosphorylation of inositol 4-phosphate (Ins-4-P) on the D5 position of the inositol ring, producing inositol 4,5-bisphosphate (Ins-4,5-P₂). PIP5KIs are predominantly cytosolic proteins that are recruited to plasma and organelle membranes through association with small GTPases such as Rho and Rac. Their lipid kinase activity is augmented by these associations and by integrin signaling. Hypertonicity increases Ins-4,5-P₂. siRNA knock down of PIP5K blocks both hypertonicity-induced increase of Ins-4,5-P₂ and cytoskeletal rearrangement. PIP5KI, being constitutively phosphorylated, is activated by Ser/Thr dephosphorylation. Hypertonicity reduces phosphorylation of PIP5K and increases its lipid kinase activity. The phosphatase inhibitor calyculin A decreases basal PIP5KI activity and blocks PIP5KI activation by hypertonicity. Thus hypertonicity induces a phosphatase that activates PIP5KI lipid kinase activity by dephosphorylating it, and the resultant increase of Ins-4,5-P₂ contributes to the ensuing cytoskeletal rearrangement.

The exact role of the cytoskeleton in osmoregulation remains speculative (77). It could function as a cell volume sensor, it could offer mechanical protection against the deleterious effects of excessive shrinkage, and it could be involved in signal transmission from osmosensors (whatever they may be) to osmoeffectors. A role in signal transmission is supported by evidence that hypertonicity induces assembly of an actin-associated protein complex containing a scaffolding protein (OSM), a GTPase (Rac), and members of a mitogen-activated protein (MAP) kinase cascade (MEKK3, MKK3, and p38) (303). p38 is a MAP kinase that contributes to signaling hypertonicity-induced activation of the osmoprotective transcription factor TonEBP/OREBP (see sect. VE). Activation of p38 by hypertonicity is inhibited if any of the components of the OSM complex is knocked out (303). These findings associate the actin cytoskeleton with a signaling molecule (p38) that contributes to osmoprotective transcription. However, the exact role of the actin cytoskeleton in the system remains to be established. Are tonicity-dependent actin cytoskeletal changes necessary to activate p38 or is the mere presence of the cytoskeleton sufficient? Also, p38 can be activated in many ways and convey many different signals. Is the OSM system the only one involved in that activation of p38 that contributes to TonEBP/OREBP activity? Is it the most important one? An added complexity is that cytoskeletal remodeling depends on p38 in MTAL cells (35), implying that there could be positive feedback.

	IV. ORGANIC OSMOLYTES ACCUMULATE IN RESPONSE TO HYPERTONICITY

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Cells often respond to osmotic shrinkage by RVI in which transporters are activated that mediate rapid uptake of inorganic ions, followed by an osmotic influx of water that restores cell volume. Many of the cellular components in Table 1 are involved in RVI. However, we will not consider RVI in detail here, referring instead to a general review (311). Although RVI increases cell volume back towards normal within minutes, the concentration of intracellular ions, and, consequently, the intracellular ionic strength remain abnormally high. This is an unhealthy state, since high ionic strength perturbs intracellular macromolecules (325).

Over a period of hours, however, compatible organic osmolytes accumulate in cells, gradually reducing the ionic strength back towards that in the normotonic state, while maintaining cell volume (Figs. 1 and 2). The benefit is that compatible organic osmolytes perturb less than do inorganic ions (325) because they stabilize proteins by interacting favorably with the protein backbone (282). The principal compatible organic osmolytes in renal medullary cells are sorbitol, glycine betaine (betaine), myo-inositol (inositol), taurine, and glycerophosphocholine (GPC) (8, 215). We previously reviewed (95) the evidence that hypertonicity increases the concentration of these organic osmolytes in renal cells both in vivo and in cell culture, and the mechanisms by which they are accumulated. To save space, we briefly summarize that earlier information and concentrate, instead, on more recent studies.

View larger version (17K): [in this window] [in a new window]   FIG. 2. High NaCl raises TonEBP/OREBP transcriptional activity via several signaling pathways, resulting in greater abundance of proteins involved in urinary concentration and in accumulation of organic osmolytes.

Sorbitol (95) is synthesized from glucose, catalyzed by aldose reductase (AR), and is metabolized to fructose, catalyzed by sorbitol dehydrogenase. Hypertonicity raises AR activity by increasing transcription of its gene, resulting in elevation of its mRNA and protein. Sorbitol dehydrogenase activity, on the other hand, is low in the renal cells that were examined and does not change significantly with tonicity. Furthermore, although hypertonicity increases AR mRNA in inner medullary collecting ducts (IMCDs) in vitro, it does not alter sorbitol dehydrogenase mRNA (106). Therefore, hypertonicity-induced increase of sorbitol apparently is entirely due to increased transcription of AR, leading to increased AR activity. TonEBP/OREBP is the transcription factor responsible for the hypertonicity-induced increase of AR. We consider osmotic regulation of TonEBP/OREBP in detail in section V.

AR is activated by other stimuli besides hypertonicity, and there are consequences of its activation besides osmoprotective synthesis of sorbitol. Although detailed consideration of other aspects of AR regulation and activity are beyond the scope of this review, it is worth noting that some of them are related to oxidative stress, which we have already seen is associated with osmotic stress (see sect. IID). For example, lipid aldehydes generated endogenously during the process of degradation of lipid peroxides contribute to cell damage associated with oxidative stress. 4-Hydroxynonenal (HNE), which is one of the most abundant and toxic lipid aldehydes, is efficiently detoxified by AR (278). AR mRNA and protein are elevated in areas of tissue destruction in inflamed arteries, colocalized with high levels of HNE, and in vitro exposure of mononuclear cells to HNE is sufficient to increase AR protein abundance (246). Similarly, HNE treatment of rat vascular smooth muscle cells induces AR mRNA (277). Oxidative stress can also activate AR posttranslationally. ROS, formed in the ischemic heart, activate AR by modifying its cysteine residues to sulfenic acids (133). Thus hypertonicity-induced increase of AR activity may affect and be affected by both the osmotic stress itself and the accompanying oxidative stress.

AR activation is not necessarily protective. AR activity is high in diabetes. This activity has been implicated in the microvascular complications of that disorder, and, as a result, there are major efforts to develop and use inhibitors of AR to minimize those complications (51). Furthermore, AR is activated during myocardial ischemic injury, and inhibitors of AR or sorbitol dehydrogenase protect rat hearts from ischemia-reperfusion injury (121). Adverse effects of increased traffic through the AR pathway have been attributed to elevation of the cytosolic NADH/NAD⁺ ratio.

B. Betaine

Betaine (95) is synthesized from choline both in liver and kidney. However, unlike sorbitol, hypertonicity does not significantly affect the rate of its synthesis. Rather, hypertonicity increases the number of transporters that carry it into the cells from the extracellular fluid. Betaine/GABA transporter (BGT1) is the osmoregulated betaine transporter (323). It couples transport of betaine to that of Cl and Na. Gradients of those ions energize the transport, providing a driving force that can concentrate betaine from micromolar levels in the plasma to millimolar levels within the cells. Hypertonicity elevates the transcription of the BGT1 gene, followed by increase in its mRNA and transport activity (302). Thus tonicity regulates betaine transporter activity by affecting BGT1 transcription. TonEBP/OREBP (see sect. V) is the transcription factor responsible for this effect.

BGT1 is regulated not only by transcription, but also by plasma membrane insertion (139). The relatively small amount of BGT1 that exists under normotonic conditions is mainly in the cytoplasm. Following hypertonicity, BGT1 is seen mainly in basolateral plasma membranes. Addition of the microtubule inhibitor nocodazole causes the additional BGT1 to remain in the cytoplasm, even following hypertonicity (139), and elevation of calcium causes internalization of BGT1, possibly mediated by PKC (138). Also, EGFP-BGT1, expressed in fibroblasts that lack endogenous BGT1, moves into plasma membranes following hypertonicity (139). Then, transport is upregulated without change in total abundance of BGT1, only its location. Interestingly, membrane insertion of existing EGFP-BGT is delayed for 3 h after tonicity increases, consistent with the need to synthesize accessory proteins for membrane insertion.

C. Inositol

Inositol (95) is synthesized by renal cells. Nevertheless, the inositol accumulated following hypertonicity is transported into the cells, not synthesized. In the absence of extracellular inositol, none is accumulated. Hypertonicity increases inositol transport by increasing the number of inositol transporters. Following its cloning, the transporter was named SMIT (sodium myo-inositol transporter)(160). SMIT couples transport of inositol to that of Na in a 1:2 ratio (109). The Na gradient energizes the transport, providing a driving force that can concentrate inositol from micromolar levels in the plasma to millimolar levels with the cells. Hypertonicity elevates the transcription of the SMIT gene, followed by increase in its mRNA and transport activity (324). Thus tonicity regulates inositol transporter activity by affecting SMIT transcription. TonEBP/OREBP (see sect. V) is the transcription factor responsible for this effect.

D. Taurine

Taurine content is regulated by tonicity in renal cells in vivo and in cell culture. Thus taurine in rat renal inner medullas is approximately twice as great when the rats are infused with high NaCl solutions (presumed to increase medullary interstitial NaCl) than when they are infused with low NaCl solutions (215). Taurine in MDCK cells doubles when tonicity is increased by adding raffinose (302). Accumulation of taurine could be due to increased synthesis or increased transport into cells. Of the two possibilities, hypertonicity-induced accumulation of taurine appears to be mainly via transport, rather than synthesis. The original evidence for transport was that hypertonicity (raffinose added) doubles taurine uptake into MDCK cells across their basolateral membranes (301). The taurine transporter that is involved was cloned by expression in Xenopus oocytes and named TauT (300). It is Na and Cl dependent, and hypertonicity increases its mRNA abundance in MDCK cells. In HepG2 cells, hypertonicity (high NaCl or sucrose) increases transcription of the TauT gene, the abundance of TauT mRNA, and the abundance of TauT protein, resulting in increased taurine uptake (126). This hypertonicity-induced increase in TauT transcription is regulated by the transcription factor TonEBP/OREBP (see sect. V) via ORE/TonE sites in the 5'-flanking region of the TauT gene (126). A possible role for Ca²⁺/calmodulin (CaM) kinase II in hypertonicity-induced activation of TauT was inferred from the action of its inhibitor, KN-93, in Caco-2 cells (261).

Taurine synthesis is from cysteine, catalyzed by the sequential actions of cysteine dioxygenase (CDO), which gives rise to cysteinesulfinate, and cysteinesulfinate decarboxylase (CSD), which decarboxylates cysteinesulfinate to hypotaurine. The synthesis is mainly in liver, from which it is released to the general circulation, a major portion being taken up by the kidneys and excreted. Although the enzymes for taurine synthesis are present in renal medullas, hypertonicity increases TauT mRNA much more in rat renal medullas than it does CDO and CSD mRNAs (22).

Taurine has many other sites of action besides the renal medulla (261). It regulates heart rhythm, contractile function, blood pressure, platelet aggregation, neuronal excitability, body temperature, learning, motor behavior, food consumption, eye sight, sperm motility, cell proliferation and viability, energy metabolism, and bile acid synthesis. At least some of these effects have been attributed to its effects on membranes and on protein phosphorylation. Thus it is conceivable that taurine that is accumulated by cells in response to hypertonicity affects them in other ways besides serving as a compatible organic osmolyte.

E. GPC

GPC (95) is abundant in cells of renal inner medullas, and it accumulates in renal cells in tissue culture in response to either high NaCl or high urea. It is synthesized from phosphatidylcholine (PC), then is broken down into choline and -glycerophosphate. The high NaCl-induced increase of GPC was studied both in isolated renal inner medullary collecting duct cells (14, 15) and in various cell cultures (156, 157, 332). In MDCK cells, high NaCl increases GPC synthesis and also reduces its degradation (156, 157). The synthesis from PC is catalyzed by a phospholipase activity that is increased by high NaCl. Similarly, phospholipase activity increases in inner medullas of rats when they are thirsted (331). Neuropathy target esterase (NTE) is a phospholipase B that catalyzes production of GPC from PC (333). In mIMCD3 cells (91), high NaCl raises NTE mRNA, beginning within 8 h, and NTE protein within 16 h. Di-isopropyl fluorophosphate (DFP), which inhibits NTE esterase activity, reduces the GPC accumulation, as does an siRNA that specifically decreases NTE protein abundance. In vivo, NTE protein expression is higher in the renal inner medulla than in the cortex. The lower renal inner medullary interstitial NaCl concentration that occurs chronically in ClCK1 –/– mice and acutely in normal mice given furosemide is associated with lower NTE mRNA and protein. Furthermore, the 20-h half-life of NTE mRNA is unaffected by high NaCl, but knock down of the transcription factor TonEBP/OREBP (see sect. V) by a specific siRNA inhibits the high NaCl-induced increase of NTE mRNA, showing that high NaCl elevates NTE mRNA through TONEBP/OREBP-mediated increase in its transcription. Transcriptional activity of TonEBP/OREBP requires binding to a specific DNA element, ORE/TonE (83, 287), whose consensus sequence is NGGAAAWDHMC(N) (83). Between bp –900 and –1 in the 5'-flanking region of NTE there are four DNA elements that fit this consensus. Thus high NaCl increases transcription of NTE, mediated by TonEBP/OREBP, and the resultant increase of NTE activity contributes to increased production and accumulation of GPC in mammalian renal cells in tissue culture and in vivo.

In addition, high NaCl also reduces degradation of GPC by decreasing the activity of GPC-choline phosphodiesterase (GPC-PDE), the enzyme that degrades GPC to choline and -glycerophosphate (15, 157, 332), and the reduced degradation of GPC contributes to its increased abundance. The GPD-PDE that is involved is Gdpd5 (unpublished observations), which is a mouse homolog of bacterial glycerol diesterases. High NaCl reduces Gdpd5 mRNA abundance. In mIMCD3 cells at 300 mosmol/kgH₂O, reducing Gdpd5 abundance with a specific siRNA increases GPC. High NaCl reduces Gdpd5 mRNA by destabilizing it. In short, high NaCl inhibits degradation of GPC by decreasing the abundance of Gdpd5, a GPC-PDE that catalyzes breakdown of GPC.

One compatible organic osmolyte can substitute for another in protecting cells from hypertonicity. For example, addition of betaine to the medium, and its resultant uptake by the cells, largely replaces the decrease in sorbitol caused by AR inhibitors and restores the viability of the cells (205). Specific reduction of one organic osmolyte generally causes a compensating increase of the others (204). This is readily understandable, since the same transcription factor, TonEBP/OREBP (see sect. V), regulates betaine and inositol transporters and the enzymes that catalyze synthesis of sorbitol and GPC. GPC is exceptional in that its degradation is also regulated (see above). It is of interest that the degradation of GPC is also affected by the abundance of the other osmolytes. Thus increasing the level of betaine and inositol in MDCK cells reduces GPC by stimulating its degradation (158). Other factors have also been reported to affect GPC. In MDCK cells, caffeine increases GPC through ryanodine-inhibitable increase of cellular calcium and activation of phospholipase A₂ (140).

	V. ACTIVATION OF THE TRANSCRIPTION FACTOR TonEBP/OREBP BY HYPERTONICITY

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TonEBP/OREBP (also named NFAT5) is a member of the Rel family of transcriptional activators, as are nuclear factor B (NFB) and nuclear factor of activated T-cells (NFAT). Each TonEBP/OREBP target gene contains in its regulatory regions at least one DNA consensus motif called an osmotic response element (ORE) (83–85, 144, 346) or tonicity-responsive enhancer (TonE) (126, 187, 200, 245, 287).

As described in section IV, TonEBP/OREBP transactivates several genes that are responsible for tonicity-dependent accumulation of organic osmolytes including sorbitol, glycine betaine, inositol, taurine, and GPC (Fig. 2) (30, 91). TonEBP/OREBP also transactivates genes coding for heat shock protein 70 (HSP70) (317), vasopressin-activated urea transporters, UT-A1 (216) and possibly UT-A2 (161), as well as aquaporin-2 (AQP2) (113, 114). As described in section XC, HSP70 is a protein chaperone and is osmoprotective against high NaCl and high urea. UT-A1 and UT-A2 recycle urea, producing high concentrations in the renal inner medulla (255), which contributes to the corticomedullary osmotic gradient necessary for concentrating urine. AQP2 increases water permeability of the renal collecting duct, which increases urine concentration. Mice that overexpress a dominant negative form of TonEBP/OREBP in renal collecting tubule cells have impaired urinary concentrating ability, probably due to reduced expression of AQP-2 and UT-A (161).

TonEBP/OREBP knock-out or dominant negative expression in vivo has major renal and extrarenal consequences. Disruption of both TonEBP/OREBP alleles in mice results in embryonic lethality (101, 176). The few surviving homozygous TonEBP/OREBP null-mice have profound, progressive atrophy of the kidney medulla, coupled with impaired activation of the TonEBP/OREBP target genes AR, BGT1, and SMIT (176). Heterozygous mice exhibit defects in adaptive immunity, evidenced by lymphoid hypocellularity and impaired antigen-specific antibody response (101). Additionally, in culture, TonEBP/OREBP –/+ lymphocytes proliferate more slowly in hypertonic, but not normotonic media (101). Mice that overexpress a dominant negative form of TonEBP/OREBP in the lens develop nuclear cataracts soon after birth (308). Developing fiber cells in these lenses exhibit a physiological state similar to that produced by hypertonicity, including DNA strand breaks, activation of p53, and induction of checkpoint kinase (308).

In the following sections we analyze how the transcriptional activity of TonEBP/OREBP is controlled and the complex network by which hypertonicity signals the activation.

A. TonEBP/OREBP mRNA and Protein Abundance

Hypertonicity increases TonEBP/OREBP mRNA in MDCK (316), HeLa (143), and mouse inner medullary collecting duct (mIMCD3) cells (39). The increase is transient, peaking at 4–12 h depending on cell type. TonEBP/OREBP mRNA rises because of a transient increase in its stability, mediated by elements within its 5'-UTR (39). In mIMCD3 cells, TonEBP/OREBP mRNA is stabilized for 6 h after addition of NaCl, as measured by determining the rate of mRNA decrease after inhibiting transcription with actinomycin D (39). Stabilization by high NaCl is sufficient to explain the increase of TonEBP/OREBP mRNA without any change in transcription (39). Following increased mRNA abundance, TonEBP/OREBP protein increases. In combination with tonicity-dependent nuclear localization, discussed below, the elevated nuclear abundance of TonEBP/OREBP increases TonEBP/OREBP protein binding to OREs. The hypertonicity-induced increase in TonEBP/OREBP protein abundance and synthesis rate is approximately equal to the increase in mRNA abundance (316). Hypertonicity does not affect the rate of TonEBP/OREBP protein degradation (316).

B. Dimerization of TonEBP/OREBP

TonEBP/OREBP is a constitutive dimer, and dimerization is required for several aspects of its hypertonicity-dependent activation. The NH₂ terminus of TonEBP/OREBP contains two dimerization domains (177). TonEBP/OREBP forms stable dimers (or higher order oligomers) in solution (178) independent of tonicity (177). Dimerization of TonEBP/OREBP is required for DNA binding (177). In binding to DNA, TonEBP/OREBP forms a homodimer that completely encircles its DNA target (284). Constitutive dimerization of TonEBP/OREBP is also required for its "proper" phosphorylation when NaCl is high (166). Dimerization also is required for TonEBP/OREBP transactivation of downstream genes (177).

C. Phosphorylation of TonEBP/OREBP

TonEBP/OREBP becomes phosphorylated on serine and tyrosine residues within 30 min of addition of NaCl (65). Possible phosphorylation sites in TonEBP/OREBP isoform "c" (1,531 amino acids) are its 216 serines, 15 tyrosines, and 111 threonines. The exact role of this tonicity-dependent TonEBP/OREBP phosphorylation remains unclear because there has been no direct identification of the amino acids that are phosphorylated. However, single and multisite phosphorylation are known to regulate other transcription factors at multiple levels, including nuclear localization, DNA binding, and transactivation.

Serine/threonine phosphorylation is involved in TonEBP/OREBP binding to its DNA element. The binding, as measured using nuclear extracts and electromobility shift assays with an ORE probe, is eliminated by treatment with calf intestinal phosphatase (CIP), a general phosphatase, and PP1, a serine/threonine phosphatase, but not by treatment with PTPase, a phosphotyrosine phosphatase (2). Nevertheless, binding of immunoprecipitated TonEBP/OREBP is not prevented by CIP, indicating that its DNA binding does not require the actual phosphorylation of TonEBP/OREBP itself (65).

Phosphorylation generally regulates transactivation 1) by recruiting coactivator proteins that contact the general transcriptional machinery, 2) as a requirement for maximal transcriptional activity, or 3) as a requirement for the formation of stable homodimer-DNA complexes. High NaCl increases phosphorylation of TonEBP/OREBP within amino acids 548–1531, as indicated by phosphatase-inhibitable decrease of migration of that fragment in gels (86). TonEBP/OREBP amino acids 872–1271 contain a transactivation domain (TAD) that is activated by high NaCl (86). Serine/threonine kinase inhibitors reduce this high NaCl-dependent transactivational activity of TonEBP/OREBP (86), which could be due either to a direct effect on the phosphorylation state of TonEBP/OREBP, itself, or it could be mediated by a signaling partner or a transcriptional cofactor. An indirect effect is plausible, since no net tonicity-dependent change in phosphorylation of the TonEBP/OREBP TAD was detected (165), while a direct effect is supported by site-directed mutation of putative phosphorylation sites (124), as discussed below.

D. TonEBP/OREBP Nuclear Localization

To bind to their cognate DNA elements, transcription factors must enter the nucleus. Proteins the size of TonEBP/OREBP are too large (>50 kDa) to diffuse freely in and out through nuclear pores. Such large molecules require a nuclear localization sequence (NLS) to mediate binding to importin. The importin-cargo complex enters the nucleus by active transport through nuclear pores. There is a similar requirement for a nuclear export sequence (NES) and binding to exportin for large proteins to exit from the nucleus. The distribution of TonEBP/OREBP in the cell is tonicity dependent. In MDCK cells, HeLa cells and Jurkat cells at normotonicity (300 mosmol/kgH₂O), TonEBP/OREBP is distributed between cytoplasm and nucleus, but at 450–500 mosmol/kgH₂O, most of the TonEBP/OREBP is in the nucleus (143, 177, 201, 296). At 135–250 mosmol/kgH₂O in MDCK or HeLa cells, TonEBP/OREBP is mostly in the cytoplasm (296, 316), correlated with a decrease in its activity (86, 316). There is indirect evidence that TonEBP/OREBP may interact with a cytoplasmic binding protein. MG-132, a 26S proteasome inhibitor, blocks tonicity-dependent nuclear localization of TonEBP/OREBP in MDCK cells (318). This calls to mind the regulation of NFB (63). In the cytoplasm, IB binds to NFB blocking NFB nuclear import. During activation of NFB, IB becomes phosphorylated, then degraded in the proteasome, releasing NFB to move into the nucleus. Although a similar system could exist for regulation of TonEBP/OREBP, no TonEBP/OREBP cytoplasmic binding protein analogous to IB has been identified. MG-132 also blocks NaCl-induced nuclear localization in HepG2 cells, but not in COS-7 cells, indicating that the effect is cell type dependent (338). Cyclosporin A has a similar effect in MDCK cells (267), consistent with calcineurin dependence of nuclear localization.

Like the native protein, a recombinant TonEBP/OREBP expressing NH₂-terminal amino acids 1–547 localizes to the nucleus when NaCl is increased, indicating that the COOH terminus is not required for translocation (338). The NH₂ terminus of TonEBP/OREBP contains a series of amino acids (amino acids 199–216 of isoform "c") that fit with a consensus bipartite NLS (143, 179) in that there are two clusters of basic amino acids in tandem. However, the NLS of TonEBP/OREBP is actually monopartite. Only the first cluster of basic amino acids is necessary for its nuclear import (296). The NH₂ terminus of TonEBP/OREBP additionally contains two motifs that function in nuclear export (296). The NES at amino acids 8–15 (isoform "c") is a CRM1-responsive protein domain. Accordingly, leptomycin B, an inhibitor of the exportin CRM1, blocks nucleocytoplasmic shuttling of TonEBP/OREBP at 300 mosmol/kgH₂O (296). Disruption of this NES increases TonEBP/OREBP in the nucleus at 300 mosmol/kgH₂O, but hypotonicity can still cause nuclear export of this mutant, indicating that this NES is not responsible for nuclear export under hypotonic conditions (296). An auxiliary export domain (AED) is found at amino acids 132–156 (isoform "c") and is functional in nuclear export of TonEBP/OREBP under both normotonic and hypotonic conditions. Deletion of the AED causes nuclear retention of TonEBP/OREBP regardless of tonicity (296). However, the AED does not affect subcellular localization when fused to green fluorescent protein, indicating it does not function independently (296).

Activation of ataxia telangiectasia-mutated (ATM) kinase by high NaCl contributes to nuclear translocation of TonEBP/OREBP (338). In cells lacking functional ATM, nuclear translocation of native TonEBP/OREBP and of a recombinant NH₂-terminal construct (amino acids 1–547, which contains the NLS as well as the NES and AED) is reduced, and reconstituting the kinase in these cells restores the translocation (338). Thus ATM contributes to high NaCl-induced nuclear translocation of TonEBP/OREBP through interaction at sites within amino acids 1–547. The effect of ATM could be direct or it could be mediated by another factor in the signaling pathway that activates TonEBP/OREBP. Since transactivation domains can affect nucleocytoplasmic shuttling (238), it is possible that that ATM also modulates translocation via sites in TonEBP/OREBP-548–1531, which contains a tonicity-dependent transactivation domain as well as putative ATM consensus phosphorylation sites (86, 124).

Other kinases such as Fyn and PI-3K class 1A, which also are known to stimulate TonEBP/OREBP-dependent transcription, do not affect NaCl-dependent nuclear localization of TonEBP/OREBP (123, 142), but, as discussed below, each of these kinases does target the hypertonicity-sensitive TonEBP/OREBP transactivation domain.

E. TonEBP/OREBP Transactivational Activity

In addition to motifs that regulate nuclear localization and DNA binding, transcription factors also contain transactivation domains (TADs), which interact with and recruit a specific assemblage of proteins that forms an enhanceosome, and with the basal transcriptional complex which includes RNA polymerase. Transactivation of target genes by transcription factors is regulated by DNA binding, by association with cofactors, by heterologous transcription factors, and by posttranslational modifications. Transactivation of target genes by TonEBP/OREBP varies directly with extracellular NaCl concentration (86). TonEBP/OREBP has a high NaCl-dependent TAD within amino acids 1039–1249 (isoform "c") (86, 165). Additional TonEBP/OREBP TADs are located within amino acids 1–76 and 1363–1476, but these are not sensitive to tonicity (165). TonEBP/OREBP also has two modulation domains (amino acids 618–820 and 889–955) that potentiate the activity of the TADs but are not independently active in stimulating transcription. One of the modulation domains is responsive to tonicity (amino acids 618–820), and all of the domains act synergistically to transactivate target genes in response to hypertonicity (165).

Many regulators contribute to tonicity-dependent increase in TonEBP/OREBP transactivation of its target genes. Each is necessary for full TonEBP/OREBP activity, but no one of them is sufficient.

cAMP-dependent kinase (PKA) is a serine-threonine kinase that regulates many cellular processes, most commonly in response to increased intracellular cAMP. However, high NaCl activates PKAc without increasing intracellular cAMP (82). Chemical inhibition of PKAc or transfection with a PKAc dominant negative construct reduces hypertonicity-induced TonEBP/OREBP transcriptional activity, transactivation activity, and resultant accumulation of AR and BGT1 mRNAs (82). Conversely, overexpression of wild-type PKAc increases TonEBP/OREBP transactivation activity (82). Also, PKAc and TonEBP/OREBP reciprocally coimmunoprecipitate each other, indicative of physical association of the proteins (82).

p38 is a mitogen-activated protein kinase (MAPK). Hypertonicity activates p38 (111), contributing to increased TonEBP/OREBP activity (142, 210, 265). Thus inhibition of p38 by chemicals (142, 210, 265) or by transfection of a dominant negative construct (142) reduces 1) hypertonicity-induced activation of TonEBP/OREBP (142, 210), 2) the increase in its transactivational activity (142), and 3) the resultant increase of mRNA abundance of its transcriptional targets, AR and BGT1 (142, 265). The pathway for activation of p38 MAPK by osmotic stress is cell type-specific and can involve different combinations of the MAPK kinases, MKK3 and MKK6, as well as autoactivation (134). Hypertonicity causes MEKK3 activation (303) by autophosphorylation (89) and also activates MKK3 and MKK6 (62, 194). p38, itself, becomes autoactivated in response to hypertonicity, promoted by association with transforming growth factor--activated protein kinase 1-binding protein (TAB1) (134). Furthermore, some of the cellular p38 is contained in a multienzyme complex, attached to the scaffolding protein, OSM. Hypertonicity activates p38 in the complex, associated with hypertonicity-induced cytoskeletal rearrangement (see sect. IIIB) (303). The complex attached to OSM also includes actin, a GTPase (Rac), MEKK3, and MKK3 (303). Overexpression of dominant negative Rac or siRNA knockdown of OSM or MEKK3 reduces the hypertonicity-induced phosphorylation and activation of p38 (303). TonEBP/OREBP activity was not addressed in these studies. Although linkage of TonEBP/OREBP activity to Rac and OSM remains conjectural, activation of MEKK3 does stimulate TonEBP/OREBP (226). Overexpression of recombinant active MEKK3 in MDCK cells increases TonEBP/OREBP transcriptional activity and the mRNA abundance of the TonEBP/OREBP target gene BGT1 (226). Additionally, siRNA-mediated depletion of native MEKK3 in HEK293 cells reduces BGT1 mRNA (226). In other experiments overexpression of constitutively active MKK3 or MKK6 did not affect TonEBP/OREBP expression or function (177), and dominant negative MKK3 did not affect TonEBP/OREBP transcriptional activity (154). Thus there is some uncertainty as to the composition of the MAPK kinase signaling module that mediates hypertonicity-dependent TonEBP/OREBP activation via p38. As is true for all other factors known to regulate TonEBP/OREBP activity, inhibition of p38 MAPK only partially reduces the hypertonicity-induced increase of TonEBP/OREBP activity (142). Activation of p38 by hypertonicity also apparently depends on the accompanying hypertonicity-induced increase of ROS (see sect. IID). Thus the antioxidant NAC prevents high NaCl-induced phosphorylation of p38 in mIMCD-K2 cells (328).

Fyn is a member of the SRC family of nonreceptor, cytoplasmic protein tyrosine kinases. Regulation of SRC kinases involves both phosphorylation and dephosphorylation. Inactive SRC kinases are constitutively phosphorylated at a COOH-terminal tyrosine. Upon dephosphorylation of the COOH-terminal tyrosine, SRC kinases undergo a conformational change that exposes the catalytic domain. Autophosphorylation of a regulatory tyrosine within the catalytic domain is required for full activation. Various SRC tyrosine kinases are involved in diverse cellular responses to hypertonicity that include RVI, reorganization of cytoskeletal proteins, and mechanotransduction (53). Several studies have investigated the roles of SRC kinases in TonEBP/OREBP activation. Herbimycin, an SRC kinase inhibitor, decreases high NaCl-induced TonEBP/OREBP-dependent transcription, transactivational activity, and upregulation of TonEBP/OREBP target gene mRNAs (86). Among SRC family kinases, Fyn and Yes are activated by high NaCl (136, 243). Several lines of evidence show that Fyn stimulates TonEBP/OREBP transactivation of its target genes (142). Fyn null cells have lower TonEBP/OREBP transcriptional activity than wild-type cells and show reduced hypertonic induction of the TonEBP/OREBP target gene AR (142). Furthermore, both pharmacological and dominant negative-based inhibition of Fyn decrease TonEBP/OREBP-dependent transcription (142). Fyn is activated by the cell shrinkage that results from hypertonicity, rather than an increase in ionic strength (136). SRC kinase activation of cell surface receptors is of interest as a possible osmotic sensor upstream of TonEBP/OREBP. Hypertonicity activates cell surface epidermal growth factor receptor (EGFR) tyrosine kinase (253), apparently dependent on the SRC kinase Yes. Hyperosmotic stress signals an oxidative stress-dependent Yes kinase activation, followed by rapid Yes/EGFR association (243). However, no link between Yes or EGFR and TonEBP/OREBP activation has, as yet, been established. Fyn, which does contribute to activation of TonEBP/OREBP (142), does not associate with EGFR during hyperosmotic stress (243).

ATM is a serine/threonine protein kinase that is activated by intermolecular autophosphorylation on serine-1981 following DNA breakage. Several lines of evidence indicate that the activating signal is change in higher-order chromatin structure caused by breakage of the phosphodiester backbone, rather than the DNA breaks per se. Once activated, ATM phosphorylates several proteins including p53, NBS1, BRCA1, and SMC1, that initiate cell cycle arrest, induce repair of DNA double-strand breaks, and enhance cell survival. As previously discussed in section IIC, high NaCl causes DNA strand breaks in cell culture and in vivo. High NaCl also deranges chromatin structure (152), presumably associated with the DNA breaks. That led to the hypothesis that high NaCl might activate ATM and stimulate TonEBP/OREBP transactivation of its target genes (124). Pharmacological inhibition of ATM reduces high NaCl-dependent increase in TonEBP/OREBP-mediated transcription and the resultant increase in BGT1 mRNA abundance (124). Additionally, reconstitution of AT cells, which do not express function ATM, with wild-type ATM increases high NaCl-dependent TonEBP/OREBP transcriptional and transactivational activity. In contrast, reconstitution with inactive ATM that is mutated at S1981 has no effect. ATM and TonEBP/OREBP reciprocally coimmunoprecipitate, and ATM is present along with TonEBP/OREBP at ORE sites in vitro, indicative of physical association (124). ATM kinase activity is necessary for full activation of TonEBP/OREBP, but it is not clear whether ATM directly phosphorylates TonEBP/OREBP or some intermediate protein. TonEBP/OREBP has three serines in putative ATM consensus phosphorylation motifs within COOH-terminal amino acids 548–1531 (124). As discussed above, high NaCl increases the activity of the TAD that resides in this region and causes a posttranslational modification consistent with increased phosphorylation (86). Mutation of serine-1247 in TonEBP/OREBP reduces its transcriptional activity in HEK293 cells, but does not in AT cells (which lack functional ATM), unless the AT cells are reconstituted with functional ATM (124). These results support ATM-mediated phosphorylation of TonEBP/OREBP. However, the conclusion remains questionable as long as there is no direct evidence linking any change in phosphorylation of a specific amino acid in TonEBP/OREBP accompanying hypertonicity-induced increase of TonEBP/OREBP transactivational activity (165). Multiple agents activate ATM without activating TonEBP/OREBP (124), including urea, UV, IR, and low NaCl (11, 124). Furthermore, high urea (293) or low NaCl (86) actually decrease TonEBP/OREBP activation. Thus ATM is necessary for full TonEBP/OREBP activity, but activation of ATM, alone, is not sufficient to induce it.

PI3Ks are intracellular lipid kinases that phosphorylate the 3'-hydroxyl moiety of phosphatidylinositol and phosphoinositides. Many different signaling proteins are activated by binding to the lipid products of PI3Ks. Three classes of PI3Ks are recognized, based on sequence homology and lipid substrate specificity. Class IA (PI3K-IA), which generates phosphatidylinositol 3,4,5-trisphosphate (PIP₃) from phosphatidylinositol 4,5-bisphosphate (PIP₂), is involved in activation of TonEBP/OREBP (78). Elevation of NaCl results in phosphorylation of tyrosine-508 of the PI3K-IA p85 regulatory subunit and activates the p110 catalytic subunit, resulting in increased production of PIP₃ (123). Knockdown of PI3K-IA by a dominant negative mutant of p85 reduces high NaCl-induced transcriptional and transactivational activity of TonEBP/OREBP, and knockdown by an siRNA against p110 reduces TonEBP/OREBP-driven transcription (123). These effects of PI3K-IA are mediated by ATM (123). The interaction between PI3K-IA and ATM is discussed below. Neither the dominant negative- or siRNA-mediated knockdown of PI3K-IA affects high NaCl-induced increase in TonEBP/OREBP protein abundance (123). As with other kinases, inhibition of PI3K-IA does not completely eliminate high NaCl-induced activation TonEBP/OREBP. It is necessary for full activation of TonEBP/OREBP, but is not in itself sufficient.

F. Interactions Between Proteins That Regulate TonEBP/OREBP

To date, we are certain of only two regulatory proteins whose actions on TonEBP/OREBP are interrelated. The effect of PI3K-IA on TonEBP/OREBP is mediated through ATM, since ATM activation, as measured by S1981 phosphorylation in response to high NaCl, is abrogated by expression of dominant negative acting PI3K-1A (123). Additionally, the effects of inhibiting PI3K-IA and ATM are equivalent and are not additive. A PI3K-1A interaction with p38 interaction also was investigated but yielded negative results, indicating that high NaCl activation of p38 is not mediated by PI3K-1A (123). Similarly, p38 and Fyn act independently, since inhibition or deficiency of one does not affect activation of the other, and their effects on TonEBP/OREBP are additive (142). Presumably, other interactions among proteins that regulate TonEBP/OREBP remain to be determined.

G. Inhibition of TonEBP/OREBP

RNA helicase A (RHA) is a nuclear RNA-associated protein that unwinds DNA and RNA. RHA inhibits activation of TonEBP/OREBP, independent of its helicase activity (59). It associates with TonEBP/OREBP in vitro and in vivo as demonstrated by reciprocal pull down. The NH₂ and COOH termini of RHA bind TonEBP/OREBP at the NH₂-terminal E'F loop, which is the region of TonEBP/OREBP that forms a secondary dimer interface involved in encirclement of DNA. The association of RHA with TonEBP/OREBP is independent of DNA binding and TonEBP/OREBP dimerization, and it does not involve any of the TonEBP/OREBP transactivation domains. Hypertonicity reduces binding of RHA to TonEBP/OREBP, and overexpression of RHA partially reduces TonEBP/OREBP transcriptional activity. Thus hypertonicity apparently decreases RHA-TonEBP/OREBP binding, which releases TonEBP/OREBP from inhibition by RHA.

H. DNA Breaks, ROS, and Cytoskeletal Perturbations as Regulators of TonEBP/OREBP

DNA breaks increase in response to high NaCl both in cell culture and in vivo (see sect. IIC). The DNA breaks, by causing change in higher-order chromatin structure, activate PI3K-IA and ATM, which initiates cell cycle arrest and chromosomal repair and promotes cell survival. PI3K-IA and ATM also stimulate TonEBP/OREBP (see sect. VE), thus linking TonEBP/OREBP activation to high NaCl-induced DNA breaks.

ROS convey signals from cytokines, hormones, and ions to downstream effectors, including transcription factors (87). High NaCl increases intracellular ROS in renal cells in culture (328, 337, 348) and in the renal medulla (337). Renal papillary ROS come from mitochondria (352), as do the NaCl-induced ROS that contribute to activation of TonEBP/OREBP (347, 348). Reduction of ROS by antioxidants decreases high NaCl-induced TonEBP/OREBP activation. The antioxidants reduce high NaCl-induced TonEBP/OREBP transcriptional activity and the mRNA abundance of its target gene BGT1. Mitochondrial ROS stimulate NaCl-induced TonEBP/OREBP transactivating activity, but do not affect its nuclear localization (347, 348). Similar to ATM activation, the increased ROS are necessary for full high NaCl-induced activation of TonEBP/OREBP, but elevation of ROS, by itself, does not activate TonEBP/OREBP (124, 348).

Actin cytoskeleton reorganization, which is induced by hypertonicity (see sect. IIIB), contributes to activation of TonEBP/OREBP. Hypertonicity induces the phosphorylation and/or redistribution of myosin, mediated by activation of the Rho and Rho/ROK pathway (69). Hypertonicity also stimulates the Rho family GTPase, Rac (68) which associates with OSM, actin, MEKK3, and MKK3 to activate p38 (303), providing a possible link to activation of TonEBP/OREBP. Another possible connection between cytoskeletal rearrangement and TonEBP/OREBP activation is through the increased mitochondrial ROS production that occurs in response to actin cytoskeleton remodeling, mediated by integrins and activation of Rac and RhoA (4, 314).

Integrins are extracellular matrix receptors that link the internal actin cytoskeleton to the extracellular matrix and thus mediate mechanotransduction. Hypertonicity induces mRNA and protein expression of the cell adhesion molecule ₁-integrin in MDCK cells (see sect. IIIB). ₁₁-Integrin is a collagen-binding receptor that is highly expressed in the kidney. In response to dehydration, inner medullas of ₁₁-integrin null mice have reduced accumulation of the organic osmolytes, sorbitol, betaine, and inositol as well as mRNA abundance of the inositol transporter SMIT and the sorbitol synthesizing enzyme AR (202). Furthermore, primary cultures of inner medullary collecting duct cells that lack ₁₁-integrin have impaired hypertonic induction of TonEBP/OREBP protein (202). Thus ₁₁-integrin is involved in hypertonicity-induced activation of TonEBP/OREBP.

	VI. ADAPTATION TO CHRONIC HYPERTONICITY

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Many of the changes in Table 1 were measured within minutes of increasing the osmolality, so they could be interpreted as transient osmoregulatory responses or as perturbations to be normalized when osmoregulation is complete. However, many of the changes also persist when the duration of the measurements is extended and even continue in cells adapted to hyperosmolality. Cells in the renal medulla are a prime example of being adapted to hyperosmolality. They survive and function despite normally being always exposed to high levels of NaCl and urea. Although cells in tissue culture are killed by acute exposure to levels of NaCl and urea far lower than those in the renal medulla (198, 257), the tissue culture cells, nevertheless, can be chronically adapted to high levels of NaCl and urea. Examples include PAP-HT25 (299), MDCK (212), and mIMCD3 cells (42, 240). Many osmoprotective responses, such as high levels of organic osmolytes (95) elevated expression of heat shock proteins (17, 260) and Na⁺-K⁺-ATPase (42) continue in adapted cells. This is somewhat surprising since the immediate effects of hypertonicity (cell shrinkage and increase in concentrations of intracellular components) are negated by RVI and accumulation of organic osmolytes (see sect. IV). That leaves the question of what the signal could be that maintains the osmoregulatory responses in adapted cells. A possibility is that the reversal of the immediate effects of hypertonicity is sufficient to keep the cells alive and functioning, but is not complete. Then, a residual small reduction of cell volume, associated with increased macromolecular crowding, and/or a residual small increase of ionic strength, might suffice to account for the continued DNA breaks (see sect. IIC) and oxidative stress (see sect. IID) and to maintain the osmoprotective responses. Until now, measurements in cell culture (212, 298) and in vivo (16) have shown more or less complete recovery of cell volume and ionic strength in adapted cells. However, the precision of those assays is not sufficient to measure small, yet possibly important, differences from cells not stressed by hypertonicity.

	VII. SENSORS AND SIGNALERS OF HYPERTONICITY

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The immediate effect of hypertonicity is decreased cell volume, accompanied by increased concentration of all intracellular components, including inorganic ions and macromolecules. There is evidence that any of these stresses can signal an osmoregulatory response. That includes cell shrinkage per se (69, 79, 98, 151, 220, 247, 251) and increased intracellular ionic strength (69, 171, 220, 298, 316). Furthermore, the cell shrinkage could act through macromolecular crowding (29, 96) and cytoskeletal perturbation (231, 303). Macromolecular crowding and ionic strength, in particular, have profound effects on biochemical processes, which could contribute to the increased ROS (see sect. IID), DNA breaks (see sect. IIC), and altered activity of kinases and other enzymes that are involved in activating TonEBP/OREBP (see sect. VC). No specific mammalian osmosensor has yet been identified. It is possible that none exists. In that case it may be the complex array of changes that we just outlined, rather than a specific sensor, that senses hypertonicity and triggers osmoregulatory responses.

	VIII. HIGH UREA

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Since urea readily permeates most cells, high urea is not generally hypertonic. Nevertheless, it directly perturbs and denatures proteins and nucleic acids (325), and it affects many cellular processes (Table 1). Tissues in which urea concentration is high generally contain methylamine organic osmolytes that counteract the perturbations (325).

A. Cellular Components and Functions Affected by High Urea

High urea increases expression and activity of transcription factors, enzymes (particularly kinases), and other signaling proteins (Table 1), but it also decreases the expression and activity of others, an effect in some cases opposite to that of hypertonicity (Table 1).

Many aspects of high urea-induced signaling are reminiscent of those observed in response to peptide mitogens (292). Multiple receptor tyrosine kinase effector pathways are activated by urea in renal medullary cells, including 1) Shc/Grb2/SOS/Ras/Raf/MAPK/(MEK)/ERK/Elk-1 (52, 341), 2) PLC-/Ins(1,4,5)P₃ (52), and 3) PI3K/Akt/p70 S6 kinase (341) pathways, as illustrated in Figure 1 of Reference 292. An initial step is epidermal growth factor (EGF) receptor transactivation through an autocrine mechanism. Enzymic cleavage by a metalloproteinase results in ectodomain shedding that liberates heparin-binding EGF as a soluble peptide agonist (343). This catalyzes the observed tyrosine phosphorylation of the adapter protein Shc (341). Shc recruits a second adapter protein, Grb2 (341). The Grb2 interaction partner and guanine nucleotide exchange factor, SOS, also becomes tyrosine phosphorylated, associated with rapid activation of the small G protein Ras (291). Subsequent events are activation of Raf (292), MEK (329), ERK (52), and the transcription factor Elk-1 (52), resulting in increased transcription of immediate-early genes, including Egr-1 (54, 56). Interestingly, mitogenesis in response to urea treatment was not observed in the mIMCD3 cells that were used for most of these studies (292). Nevertheless, indices of proliferation in response to urea treatment have been observed in other urea-responsive renal epithelial cell lines such as MDCK, LLC-PK₁, and NRK-52E (55, 56, 174). Thus this urea-induced mitogenic signaling pathway has been thoroughly documented, but its physiological role remains unclear.

Similarly, in microarray studies of mIMCD3 cells, the response to high urea was closer to that of the growth factor EGF than to hypertonic stress (294). High urea decreases abundance of many mRNAs. Approximately 6% of the 12,000 transcripts were downregulated, whereas only 0.5–0.8% were upregulated. mRNA expression of only 21 genes was upregulated threefold or more (294). Prominent among them was a large increase (27-fold) of activating transcription factor 3 (ATF3) mRNA that was accompanied by a large increase in its protein abundance (294). ATF3 is a member of the mammalian activation transcription factor/cAMP responsive element-binding (CREB) protein family of transcription factors. It is induced by a large number of other stresses, including endoplasmic reticulum stress and amino acid starvation (130), and it contributes to the accompanying induction of Gadd153. High urea also increases expression of Gadd153 (340), as well as of the cytoprotective enzyme heme oxygenase-1 (38, 290), both dependent on urea-induced oxidative stress (290, 340) and on Ras (291).

B. Counteraction of Urea Effects by GPC

Urea is sufficiently high in renal medullas to perturb macromolecules (see introduction to sect. VIII), yet the cells survive and function. Methylamine organic osmolytes protect cells from high urea by counteracting its effects (325). Animal tissues containing high levels of urea generally also contain high levels of methylamines (325). Most studies of counteraction have involved examining the effects of combinations of urea and methylamines on structure and function of macromolecules in vitro. Optimal counteraction generally occurs when the molar ratio of urea to methylamine is 2:1, which is the ratio in many tissues (325). The methylamine organic osmolytes in renal medullas are betaine and GPC (95). Both can counteract the effects of urea (31, 33). Nevertheless, although high urea increases GPC in tissue culture, it reduces betaine (95). Also, the level of GPC in renal medullas correlates with the level of urea, but betaine does not (95). Thus, although both betaine and GPC effectively counteract the effects of urea, renal cells apparently prefer GPC (Fig. 3). The reason for this preference is speculative. In so far as accumulation of GPC in response to high urea involves reducing its degradation (157, 332), the energetic cost may be less than that of transporting betaine into the cells against a high concentration gradient. Alternatively, GPC might have some unrecognized additional beneficial effect that betaine lacks.

View larger version (8K): [in this window] [in a new window]   FIG. 3. High urea denatures proteins, but also inhibits GPC-PDE, an enzyme that catalyzes degradation of glycerophosphocholine (GPC). The resulting increase of GPC counteracts denaturation of the proteins.

C. Mutually Protective Effects of High Urea and High NaCl

A high concentration of either NaCl or urea can perturb and even kill cells (see sect. IIB). However, their effects are not additive, and one can protect against the other. There are numerous examples of such mutual protection, as follows.

Adding either NaCl or urea to a total osmolality of 700 mosmol/kgH₂O kills >90% of the mIMCD3 cells by apoptosis within 24 h. However, the cells are more tolerant of NaCl and urea when they are added in combination than when they are added alone; 40% of cells survive equiosmolal addition of NaCl plus urea to 900 mosmol/kgH₂O. Added NaCl causes a rapid decrease in RNA and protein synthesis, which parallels the later apoptosis, whereas lethal doses of urea have little or no effect on these biosynthetic processes. The combination of NaCl plus urea results in an increase of Hsp70 expression that correlates with survival. Thus high NaCl and urea activate independent and complementary cellular programs that in combination enhance osmotolerance of mIMCD3 cells (257). In somewhat similar studies (198), elevating NaCl or urea reduced the number of proliferating mIMCD3 cells by slowing the transit through the S phase, by cell cycle delay in the G₂M and G₁, and by inducing apoptotic cell death. The effects were not additive in that concentrations of NaCl and urea elevated in combination had no greater effect than the individual concentrations added alone.

High urea protects against high NaCl and vice versa, but different mechanisms seem to be involved. Pretreatment with urea protects mIMCD3 cells from high NaCl-induced apoptosis (339). The signals induced by high urea are similar to those induced by peptide mitogens, and urea is mitogenic, at least in some cell lines (see sect. VIIIA). Importantly, the peptide mitogens EGF and insulin-like growth factor have protective effects comparable to that of urea. Also, urea activates EGF receptor kinase (see sect. VA), and preventing this action negates the protective effect of urea against high NaCl (343). Thus the protective effect of high urea against high NaCl apparently is related to activation of the EGF receptor by urea.

Pretreatment with NaCl protects MDCK cells from high urea-induced apoptosis. The experiments were on cells conditioned by increasing osmolality from 290 to 600 mosmol/kgH₂O by adding NaCl. Upon subsequent exposure to an additional 600 mM urea in the medium for 24 h, 90% of the osmotically conditioned cells but only 15% of nonconditioned cells survive. The effect was attributed to protection against urea by HSP72, which is strongly induced by high NaCl, but much less so by high urea (219). In the absence of hypertonic stress, survival rates of MDCK cells exposed for 24 h to 600 mM urea is improved from 14 to 43% by inducible overexpression of HSP72 (218). Thus the protective effect of high NaCl against high urea apparently is related to induction of HSP72 by high NaCl.

	IX. WHY IS OSMOTIC TOLERANCE OF RENAL MEDULLARY CELLS GREATER IN VIVO THAN IN CULTURE?

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Renal inner medullary cells survive and function in vivo despite high interstitial NaCl and urea that produce a total osmolality of 600–1,700 mosmol/kgH₂O or more, depending on the species and conditions (see sect. I). In contrast, much smaller changes kill inner medullary cells in tissue culture. mIMCD3 cells do not survive an acute increase of NaCl or urea much above 600 mosmol/kgH₂O (198, 257). The same is true of other continuous cell lines, which is why so many studies of osmotic stress do not raise osmolality above 600 mosmol/kgH₂O (Table 1), and why, in studies that do, there may be a serious question of cell viability. In what follows we discuss factors that provide the greater osmotic tolerance in vivo.

One factor is rate of proliferation and the associated DNA replication. Proliferation is rapid in continuous lines but is very slow in vivo (335). Proliferation in vivo was measured by counting cells that express proliferating cell nuclear antigen (PCNA). It is very low in the rat renal inner medulla (335). The effect of proliferation on osmotic tolerance was studied by comparing a continuous line of renal inner medullary cells (mIMCD3) to early passage cells (p2mIME). Even when they are confluent, 20% of mIMCD3 cells are in the S phase, replicating DNA, whereas only 5% of p2mIME cells are (335). This is pertinent, since cells in S phase are particularly susceptible to the apoptosis caused by high NaCl (72). Starting from a baseline of 300 mosmol/kgH₂O, few mIMCD3 cells survive acute increase of NaCl or urea above 600 mosmol/kgH₂O. In contrast, most p2mIME cells survive NaCl added to 1,100 mosmol/kgH₂O and of urea added to 700 mosmol/kgH₂O. Similarly, when NaCl and urea are added in combination, mIMCD3 cells have increased apoptosis and decreased survival above 900 mosmol/kgH₂O, whereas p2mIME cells survive well up to 1,300 mosmol/kgH₂O; 1,300 mosmol/kgH₂O is still less than inner medullary cells tolerate in vivo, which suggested the existence of other factors.

Possible additional factors that are present in vivo and might account for the greater osmotic tolerance were tested in p2mIME cells (40). Several of the factors did not affect their osmotic tolerance. The ineffective factors include hormones (insulin-like growth factor I, EGF, and deamino-8-D-arginine vasopressin), growth on porous supports, and presence of matrix proteins (collagen I, collagen IV, fibronectin, laminin, or fibrillar collagen I). The one factor that does make a major difference is the time course of the osmotic change. Greater survival had previously been observed following a slow increase in osmolality. Thus increasing NaCl by 50 mosmol/kgH₂O from passage to passage conditions mIMCD3 cells to proliferate at 900 mosmol/kgH₂O, which is higher than if the change is made acutely (42). Also, an initial small elevation of NaCl enhances survival of mIMCD3 after a later larger increase (168). However, the time course in these experiments was days or weeks, whereas inner medullary cells in vivo survive large increases of osmolality over a period of hours. During those hours, however, the increase is gradual, not an abrupt single step as in most tissue culture experiments. Under normal conditions, NaCl and urea change more or less in concert in renal inner medullary interstitial fluid. When osmolality is increased from 640 to 1,640 mosmol/kgH₂O by addition of NaCl and urea in a single step, only 30% of p2mIME cells survive for 24 h. However, when the same increase is made linearly over 20 h, 89% of the cells remain viable 24 h later. Thus gradual changes in osmolality allow cells to survive much greater changes than do the step changes routinely used in cell culture experiments. A possible factor in this difference is that when the rate of change of osmolality is slow enough, isovolumetric regulation (IVR) occurs, i.e., no measurable change occurs in cell volume, despite large cumulative changes in osmolality (175, 208). In contrast, a step increase stresses cells more by rapidly shrinking them (175).

The more gradual, linear increase in osmolality is accompanied by a greater increase in mRNA expression of many genes known to be osmoprotective (38). For example, when with p2mIME cells NaCl and urea are increased in combination from total osmolality of 640 to 1,640 mosmol/kgH₂O, a linear increase raises TonEBP/OREBP and AR mRNA, but a step increase reduces them. Heat shock proteins 70.1 (HSP70.1) and HSP70.3 increase more with a linear than a step increase, and osmotic stress protein 94 (OSP94) and heme oxygenase-1 (HO-1) increase with a linear, but decrease with a step increase. mRNAs for known urea-responsive proteins, GADD153 and Egr-1, increase with both a step and linear increase. A step increase in urea alone reduces mRNAs, similar to the combination of NaCl and urea, but a step increase in NaCl alone does not. Thus the poorer survival of p2mIME cells with a step than with linear increase in NaCl and urea is accounted for, at least in part, by urea-induced suppression of protective genes, particularly HSP70.

	X. OSMOPROTECTION BY GENERAL STRESS PROTEINS IN CELL CULTURE AND IN VIVO

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In cell culture, hyperosmolality increases expression and/or activity of many general stress proteins. Examples are Gadd45, p53, and various heat shock proteins (Table 1). In what follows, we consider the evidence (or lack of it) that these general stress proteins contribute osmotic tolerance to renal inner medullary cells in tissue culture and also in vivo. The distinction between tissue culture and in vivo is pertinent because the conditions used for routine culture of cell lines differ in several critical ways from conditions in the renal inner medulla in vivo, including absolute level of osmolality and rapidity with which osmolality changes. Also, the rates of cellular proliferation differ markedly (see sect. IX). To determine the significance of hyperosmolality-induced changes of expression in cell culture, the routine conditions of cell culture can be modified to be more like those in vivo and, most important, the conclusions can be actually tested in vivo.

A. Gadd45

Acute elevation of NaCl increases mRNA and protein abundance of the growth arrest- and DNA damage-inducible protein Gadd45 in mIMCD3 cells (46, 155). Gadd45 was originally identified as a transcript that increases following numerous different stresses. Later, two additional Gadd45 genes were identified. The three Gadd45 genes are now called Gadd45a (the original Gadd45), Gadd45b, and Gadd45g. The Gadd45 proteins are multifunctional molecules with important roles in cell differentiation, apoptosis, negative growth control, DNA repair, genome stability, immunity, and cell survival. Possible consequences of their induction by high NaCl were initially examined by overexpressing recombinant Gadd45a, Gadd45b, or Gadd45g in mIMCD3 cells. Following acute increase of NaCl to a moderate level, each overexpressed Gadd inhibits mitosis and promotes G₂/M arrest, and, in addition, Gadd45g overexpression strongly potentiates apoptosis (186). At a higher level of NaCl, Gadd45a/b overexpression transiently increases the early indicators of apoptosis, but inhibits the later stages. From these observations, it was concluded that increased abundance of Gadd45 isoforms, when NaCl is high, contributes to low mitotic index and protection of genomic integrity in cells of the renal inner medulla (186).

However, in these studies the baseline osmolality was 300 mosmol/kgH₂O, whereas the lowest normal osmolality of renal medullary interstitial fluid is 600 mosmol/kgH₂O. Also, mIMCD3 cells are immortalized by expression of SV40, so they proliferate rapidly and continue to proliferate even after they become confluent, whereas renal inner medullary cells proliferate very slowly in vivo. When, in subsequent studies, these differences were taken into account, no effect of the Gadd45s on osmotic tolerance was found. The later studies used early passage mouse inner medullary epithelial (p2mIME) cells instead of mIMCD3. p2mIME cells tolerate much higher osmolality than mIMCD3 cells and, not being immortalized, proliferate more slowly (335). With the use of p2mIME cells (37), elevation of the baseline osmolality of 640 mosmol/kgH₂O to 1,040–1,640 mosmol/kgH₂O by adding NaCl and/or urea increases both Gadd45b and Gadd45g mRNA, but lowers Gadd45a mRNA. Thus, under conditions similar to those in the inner medulla, Gadd45a does not respond to elevations of NaCl or urea, but Gadd45b and Gadd45g do. Even for Gadd45b and Gadd45g, however, their expression was not found to affect osmotic tolerance of the cells. p2mIME cells from Gadd45b^–/–, Gadd45g^–/–, and Gadd45bg^–/– mice survive elevation of NaCl (or urea) essentially as well as do wild-type cells. Furthermore, the morphology of inner medullas from Gadd45abg^–/– mice is normal, and they produce concentrated urine. Thus, although overexpression of Gadd45s affects osmotically stressed mIMCD3 cells, their absence does not seem to affect osmotic tolerance of slowly proliferating p2mIME cells or to perturb inner medullary epithelial cells in vivo.

B. p53

p53 is best known as a tumor suppressor, but it has numerous additional functions. Accumulation and activation of p53 are promoted by DNA-damaging agents, which result in cell cycle delay during which the DNA is repaired. When the DNA is not successfully repaired, p53 activity can cause apoptosis. High NaCl increases the number of DNA strand breaks (see sect. IIC) and increases p53 protein abundance, phosphorylation on S15, and transcriptional activity (71). High NaCl is most harmful to cells when they are replicating DNA in the S phase of their cell cycle (72). In agreement with the results with other stress responses, accumulation and activation of p53 in mIMCD3 cells restrict DNA replication and reduce high NaCl-induced apoptosis in mIMCD3 cells (71, 72). The result is similar in p2mIME cells. After they become confluent, p2mIME cells from p53^–/– mice proliferate and replicate DNA more rapidly than those from p53^+/+ mice, resulting in less tolerance for high NaCl (37).

Since the increase in p53 activity that occurs in mIMCD3 cells, when NaCl is elevated, protects the cells from apoptosis (71, 72), it seemed likely that p53 might also be important for osmotic tolerance of renal inner medullary cells in vivo. However, this does not seem to be the case. Urinary concentrating ability of p53^–/– mice is normal, as is the histology of their inner medullas (37). Therefore, although p53 expression is important for osmotic tolerance of mIMCD3 cells and even of p2mIME cells that are studied under conditions similar to those in the renal inner medulla, lack of p53 apparently is not deleterious to renal inner medullary cells in vivo.

C. Heat Shock Proteins

Heat shock proteins (HSPs) are a group of highly conserved proteins that are expressed constitutively and/or induced by stress. Constitutively expressed HSPs participate in protein folding and assembly, elimination of misfolded proteins, and stabilization of newly synthesized protein in various intracellular compartments. Expression of inducible HSPs is evoked by various stresses, many of which denature proteins. The HSPs enhance cellular survival and aid cellular recovery by acting as molecular chaperones that limit or correct damage to proteins. Also, HSPs prevent apoptosis by inhibition of JNK activation, prevention of cytochrome c release from mitochondria, and disruption of apoptosomes. HSPs are abundant in renal medullas. High NaCl increases expression of HSPs, protecting cells from damaging effects of high urea (218, 219).

The abundance of several of the inducible HSPs increases along the corticomedullary osmotic gradient, including HSP25, B-crystallin, HSP70, HSP110, and Osp90 (18, 258). Since hypertonicity increases expression of these heat shock proteins in cell culture (Table 1), and dehydration increases inner medullary HSP110 and Osp94 (258), high NaCl in the renal inner medullary interstitial fluid likely accounts for the great abundance of HSPs there.

Activity of TonEBP/OREBP mediates hypertonicity-induced increase in expression of one of the isoforms of HSP70. In mice there are two HSP70 genes that have identical open reading frames but that differ in their regulation. The HSP70.1 gene contains ORE/TonEs in its 5'-flanking region, and its expression is increased both by hypertonicity and heat shock in mIMCD3 (317), mouse embryo fibroblast (MEF) (272), and BALB/c3T3 (288) cells and by high NaCl in p2mIME (second passage mouse renal inner medullary) cells (38). HSP70.3 does not contain ORE/TonEs, and its expression is increased by heat, but not by hypertonicity, in mIMCD3 (317), MEF (272), and BALB/c3T3 (288) cell lines. In contrast, high NaCl does increase HSP70.3 expression in p2mIME cells (38), which apparently is independent of TonEBP/OREBP. Expression of both hsp70-2 (the human homolog of HSP70.1) and hsp70-1 (the human homolog of HSP70.3) is increased by heat, but only hsp70-2 is increased by hypertonicity in HEK293T and human embryonic fibroblast cells (38). The human hsp70-2 promoter region contains three ORE sites, and mutation of one of these ORE sites (at –135) completely abolishes induction by hypertonicity.

HSP70.1 expression helps cells survive high NaCl both in culture and in vivo. Embryonic fibroblasts from hsp70.1–/– mice have reduced tolerance for high NaCl. After preliminary treatment with elevated NaCl (100 mM added), hsp70.1+/+ MEFs survive a subsequent addition of 600 mM of NaCl, but no hsp70.1–/– cells survive under the same conditions (272). High NaCl elevates caspase-9 and caspase-3 activity in the hsp70.1–/– cells and kills them by apoptosis (164). Renal inner medullary cells of the hsp70.1–/– mice are also more susceptible to salt stress in vivo. The mice were given drinking water that contained 3% NaCl for 30 days. Under those conditions, the renal medullas of hsp70.1–/– mice contain many apoptotic (TUNEL-positive) cells, whereas wild-type mice have only a few (272).

D. Heme oxygenase 1

Heme oxygenase 1 (HO-1) is induced by diverse stresses. It protects cells by its antioxidant, antiapoptotic, and anti-inflammatory actions (206). In the kidney, there is a gradient of HO-1 mRNA and protein, highest in the renal inner medulla and lowest in the renal cortex (351). HO-1 is induced by either high NaCl or urea in mIMCD3 (290) and p2mIME cells (38). Inhibition of HO-1 by the HO-1 inhibitor ZnDPBG reduces tolerance of mIMCD3 cells for high NaCl, but does not affect tolerance for high urea. A step increase of NaCl and urea in combination reduces HO-1 mRNA in p2mIME cells, but a linear increase raises HO-1 mRNA (38). This difference in HO-1 expression could contribute to the greater tolerance of p2mIME cells for a gradual increase than for a step increase of NaCl and urea in combination.

	XI. SUMMARY AND CONCLUSIONS

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A number of osmoprotective mechanisms, including increased abundance of compatible and counteracting organic osmolytes (see sects. V and VIIIB) and of heat shock proteins (see sect. XC), enable cells to survive and function during hyperosmotic stress that otherwise would kill them. The renal inner medulla provides a striking example. Its interstitial fluid normally contains lethally high levels of salt and urea (see sect. I), yet its cells seem healthy as long as the osmoprotective mechanisms are intact. Nevertheless, despite this apparent health, cells surviving osmotic stress still differ in many respects from when they are exposed to the much lower extracellular salt and urea levels that exist elsewhere in the body. In fact, cells adapted to osmotic stress contain lesions that in other settings would be regarded as pathological. The lesions include DNA strand breaks (see sect. IIC), oxidation of DNA bases and proteins (see sect. IID), and cytoskeletal disturbances (see sect. IIIB). In the setting of hyperosmolality, however, these seemingly pathological lesions are actually protective in that they serve as sensors that signal activation of the osmoprotective transcription factor TonEBP/OREBP (see sect. VH). It is important to recognize that when cells adapt to hyperosmolality, they enter a new state in which there are multiple changes. Many changes are already recognized (Table 1), but there are probably others that have not yet been discovered. The complexity of the adapted state is reflected in the multiple pathways that signal osmoprotection (see sect. V). Research in the past 20 years has revealed the complexity of osmotic adaptation. However, much more remains to be discovered, which provides a compelling challenge for the future.

	GRANTS

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This research was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute at the National Institutes of Health.

	ACKNOWLEDGMENTS

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Address for reprint requests and other correspondence: M. B. Burg, NIH, Bethesda, MD 20892-1603 (e-mail: maurice_burg{at}nih.gov).

	REFERENCES

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1. Abe J, Kusuhara M, Ulevitch RJ, Berk BC, Lee JD. Big mitogen-activated protein kinase 1 (BMK1) is a redox-sensitive kinase. J Biol Chem 271: 16586–16590, 1996.[Abstract/Free Full Text]
2. Aida K, Tawata M, Ikegishi Y, Onaya T. Induction of rat aldose reductase gene transcription is mediated through the cis-element, osmotic response element (ORE): increased synthesis and/or activation by phosphorylation of ORE-binding protein is a key step. Endocrinology 140: 609–617, 1999.[Abstract/Free Full Text]
3. Alfieri RR, Bonelli MA, Cavazzoni A, Briggotti M, Fumarola C, Sestili P, Mozzoni P, DEPG, Mutti A, Carnicelli D, Vacondio F, Silva C, Borghetti AF, Wheeler KP, Petronini PG. Creatine as a compatible osmolyte in muscle cells exposed to hypertonic stress. J Physiol 576: 391–401, 2006.[Abstract/Free Full Text]
4. Ali MH, Pearlstein DP, Mathieu CE, Schumacker PT. Mitochondrial requirement for endothelial responses to cyclic strain: implications for mechanotransduction. Am J Physiol Lung Cell Mol Physiol 287: L486–L496, 2004.[Abstract/Free Full Text]
5. Allemand E, Guil S, Myers M, Moscat J, Caceres JF, Krainer AR. Regulation of heterogenous nuclear ribonucleoprotein A1 transport by phosphorylation in cells stressed by osmotic shock. Proc Natl Acad Sci USA 102: 3605–3610, 2005.[Abstract/Free Full Text]
6. Ambuhl P, Amemiya M, Preisig PA, Moe OW, Alpern RJ. Chronic hyperosmolality increases NHE3 activity in OKP cells. J Clin Invest 101: 170–177, 1998.[Web of Science][Medline]
7. Arima H, Yamamoto N, Sobue K, Umenishi F, Tada T, Katsuya H, Asai K. Hyperosmolar mannitol simulates expression of aquaporins 4 and 9 through a p38 mitogen-activated protein kinase-dependent pathway in rat astrocytes. J Biol Chem 278: 44525–44534, 2003.[Abstract/Free Full Text]
8. Bagnasco S, Balaban R, Fales HM, Yang YM, Burg M. Predominant osmotically active organic solutes in rat and rabbit renal medullas. J Biol Chem 261: 5872–5877, 1986.[Abstract/Free Full Text]
9. Bagnasco S, Good D, Balaban R, Burg M. Lactate production in isolated segments of the rat nephron. Am J Physiol Renal Fluid Electrolyte Physiol 248: F522–F526, 1985.[Abstract/Free Full Text]
10. Bagnasco SM, Uchida S, Balaban RS, Kador PF, Burg MB. Induction of aldose reductase and sorbitol in renal inner medullary cells by elevated extracellular NaCl. Proc Natl Acad Sci USA 84: 1718–1720, 1987.[Abstract/Free Full Text]
11. Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421: 499–506, 2003.[CrossRef][Medline]
12. Barros LF, Barnes K, Ingram JC, Castro J, Porras OH, Baldwin SA. Hyperosmotic shock induces both activation and translocation of glucose transporters in mammalian cells. Pflügers Arch 442: 614–621, 2001.[CrossRef][Web of Science][Medline]
13. Baudouin-Legros M, Brouillard F, Cougnon M, Tondelier D, Leclerc T, Edelman A. Modulation of CFTR gene expression in HT-29 cells by extracellular hyperosmolarity. Am J Physiol Cell Physiol 278: C49–C56, 2000.[Abstract/Free Full Text]
14. Bauernschmitt HG, Kinne RK. Metabolism of the "organic osmolyte" glycerophosphorylcholine in isolated rat inner medullary collecting duct cells. I. Pathways for synthesis and degradation. Biochim Biophys Acta 1148: 331–341, 1993.[Medline]
15. Bauernschmitt HG, Kinne RK. Metabolism of the "organic osmolyte" glycerophosphorylcholine in isolated rat inner medullary collecting duct cells. II. Regulation by extracellular osmolality. Biochim Biophys Acta 1150: 25–34, 1993.[Medline]
16. Beck F, Dorge A, Rick R, Thurau K. Intra- and extracellular element concentrations of rat renal papilla in antidiuresis. Kidney Int 25: 397–403, 1984.[Web of Science][Medline]
17. Beck FX, Burger-Kentischer A, Muller E. Cellular response to osmotic stress in the renal medulla. Pflügers Arch 436: 814–827, 1998.[CrossRef][Web of Science][Medline]
18. Beck FX, Neuhofer W, Muller E. Molecular chaperones in the kidney: distribution, putative roles, regulation. Am J Physiol Renal Physiol 279: F203–F215, 2000.[Abstract/Free Full Text]
19. Bedford JJ, Bagnasco SM, Kador PF, Harris HW Jr, Burg MB. Characterization and purification of a mammalian osmoregulatory protein, aldose reductase, induced in renal medullary cells by high extracellular NaCl. J Biol Chem 262: 14255–14259, 1987.[Abstract/Free Full Text]
20. Bell LM, Leong ML, Kim B, Wang E, Park J, Hemmings BA, Firestone GL. Hyperosmotic stress stimulates promoter activity and regulates cellular utilization of the serum- and glucocorticoid-inducible protein kinase (Sgk) by a p38 MAPK-dependent pathway. J Biol Chem 275: 25262–25272, 2000.[Abstract/Free Full Text]
21. Berl T, Siriwardana G, Ao L, Butterfield LM, Heasley LE. Multiple mitogen-activated protein kinases are regulated by hyperosmolality in mouse IMCD cells. Am J Physiol Renal Fluid Electrolyte Physiol 272: F305–F311, 1997.[Abstract/Free Full Text]
22. Bitoun M, Levillain O, Tappaz M. Gene expression of the taurine transporter and taurine biosynthetic enzymes in rat kidney after antidiuresis and salt loading. Pflügers Arch 442: 87–95, 2001.[CrossRef][Web of Science][Medline]
23. Bode JG, Gatsios P, Ludwig S, Rapp UR, Haussinger D, Heinrich PC, Graeve L. The mitogen-activated protein (MAP) kinase p38 and its upstream activator MAP kinase kinase 6 are involved in the activation of signal transducer and activator of transcription by hyperosmolarity. J Biol Chem 274: 30222–30227, 1999.[Abstract/Free Full Text]
24. Bortner CD, Cidlowski JA. A necessary role for cell shrinkage in apoptosis. Biochem Pharmacol 56: 1549–1559, 1998.[CrossRef][Web of Science][Medline]
25. Bortner CD, Cidlowski JA. Absence of volume regulatory mechanisms contributes to the rapid activation of apoptosis in thymocytes. Am J Physiol Cell Physiol 271: C950–C961, 1996.[Abstract/Free Full Text]
26. Brigotti M, Petronini PG, Carnicelli D, Alfieri RR, Bonelli MA, Borghetti AF, Wheeler KP. Effects of osmolarity, ions and compatible osmolytes on cell-free protein synthesis. Biochem J 369: 369–374, 2003.[CrossRef][Web of Science][Medline]
27. Bulavin DV, Amundson SA, Fornace AJ. p38 and Chk1 kinases: different conductors for the G(2)/M checkpoint symphony. Curr Opin Genet Dev 12: 92–97, 2002.[CrossRef][Web of Science][Medline]
28. Bulavin DV, Higashimoto Y, Popoff IJ, Gaarde WA, Basrur V, Potapova O, Appella E, Fornace AJ Jr. Initiation of a G₂/M checkpoint after ultraviolet radiation requires p38 kinase. Nature 411: 102–107, 2001.[CrossRef][Medline]
29. Burg MB. Macromolecular crowding as a cell volume sensor. Cell Physiol Biochem 10: 251–256, 2000.[CrossRef][Web of Science][Medline]
30. Burg MB, Kwon ED, Kultz D. Regulation of gene expression by hypertonicity. Annu Rev Physiol 59: 437–455, 1997.[CrossRef][Web of Science][Medline]
31. Burg MB, Kwon ED, Peters EM. Glycerophosphocholine and betaine counteract the effect of urea on pyruvate kinase. Kidney Int Suppl 57: S100–S104, 1996.[CrossRef][Medline]
32. Burg MB, Peters EM. Urea and methylamines have similar effects on aldose reductase activity. Am J Physiol Renal Physiol 273: F1048–F1053, 1997.[Abstract/Free Full Text]
33. Burg MB, Peters EM. Effects of glycine betaine and glycerophosphocholine on thermal stability of ribonuclease. Am J Physiol Renal Physiol 274: F762–F765, 1998.[Abstract/Free Full Text]
34. Burg MB, Peters EM, Bohren KM, Gabbay KH. Factors affecting counteraction by methylamines of urea effects on aldose reductase. Proc Natl Acad Sci USA 96: 6517–6522, 1999.[Abstract/Free Full Text]
35. Bustamante M, Roger F, Bochaton-Piallat ML, Gabbiani G, Martin PY, Feraille E. Regulatory volume increase is associated with p38 kinase-dependent actin cytoskeleton remodeling in rat kidney MTAL. Am J Physiol Renal Physiol 285: F336–F347, 2003.[Abstract/Free Full Text]
36. Cai Q, Dmitrieva NI, Ferraris JD, Brooks HL, van Balkom BW, Burg M. Pax2 expression occurs in renal medullary epithelial cells in vivo and in cell culture, is osmoregulated, promotes osmotic tolerance. Proc Natl Acad Sci USA 102: 503–508, 2005.[Abstract/Free Full Text]
37. Cai Q, Dmitrieva NI, Ferraris JD, Michea LF, Salvador JM, Hollander MC, Fornace AJ Jr, Fenton RA, Burg MB. Effects of expression of p53 and Gadd45 on osmotic tolerance of renal inner medullary cells. Am J Physiol Renal Physiol 291: F341–F349, 2006.[Abstract/Free Full Text]
38. Cai Q, Ferraris JD, Burg MB. Greater tolerance of renal medullary cells for a slow increase in osmolality is associated with enhanced expression of HSP70 and other osmoprotective genes. Am J Physiol Renal Physiol 286: F58–F67, 2004.[Abstract/Free Full Text]
39. Cai Q, Ferraris JD, Burg MB. High NaCl increases TonEBP/OREBP mRNA and protein by stabilizing its mRNA. Am J Physiol Renal Physiol 289: F803–F807, 2005.[Abstract/Free Full Text]
40. Cai Q, Michea L, Andrews P, Zhang Z, Rocha G, Dmitrieva N, Burg MB. Rate of increase of osmolality determines osmotic tolerance of mouse inner medullary epithelial cells. Am J Physiol Renal Physiol 283: F792–F798, 2002.[Abstract/Free Full Text]
41. Capasso JM, Rivard C, Berl T. The expression of the gamma subunit of Na-K-ATPase is regulated by osmolality via C-terminal Jun kinase and phosphatidylinositol 3-kinase-dependent mechanisms. Proc Natl Acad Sci USA 98: 13414–13419, 2001.[Abstract/Free Full Text]
42. Capasso JM, Rivard CJ, Berl T. Long-term adaptation of renal cells to hypertonicity: role of MAP kinases and Na-K-ATPase. Am J Physiol Renal Physiol 280: F768–F776, 2001.[Abstract/Free Full Text]
43. Capasso JM, Rivard CJ, Enomoto LM, Berl T. Chloride, not sodium, stimulates expression of the gamma subunit of Na/K-ATPase and activates JNK in response to hypertonicity in mouse IMCD3 cells. Proc Natl Acad Sci USA 100: 6428–6433, 2003.[Abstract/Free Full Text]
44. Caruccio L, Bae S, Liu AY, Chen KY. The heat-shock transcription factor HSF1 is rapidly activated by either hyper- or hypo-osmotic stress in mammalian cells. Biochem J 327: 341–347, 1997.[Web of Science][Medline]
45. Celeste A, Petersen S, Romanienko PJ, Fernandez-Capetillo O, Chen HT, Sedelnikova OA, Reina-San-Martin B, Coppola V, Meffre E, Difilippantonio MJ, Redon C, Pilch DR, Olaru A, Eckhaus M, Camerini-Otero RD, Tessarollo L, Livak F, Manova K, Bonner WM, Nussenzweig MC, Nussenzweig A. Genomic instability in mice lacking histone H2AX. Science 296: 922–927, 2002.[Abstract/Free Full Text]
46. Chakravarty D, Cai Q, Ferraris JD, Michea L, Burg MB, Kultz D. Three GADD45 isoforms contribute to hypertonic stress phenotype of murine renal inner medullary cells. Am J Physiol Renal Physiol 283: F1020–F1029, 2002.[Abstract/Free Full Text]
47. Chan MM, Chmura K, Chan ED. Increased NaCl-induced interleukin-8 production by human bronchial epithelial cells is enhanced by the DeltaF508/W1282X mutation of the cystic fibrosis transmembrane conductance regulator gene. Cytokine 33: 309–316, 2006.[CrossRef][Web of Science][Medline]
48. Chen D, Elmendorf JS, Olson AL, Li X, Earp HS, Pessin JE. Osmotic shock stimulates GLUT4 translocation in 3T3L1 adipocytes by a novel tyrosine kinase pathway. J Biol Chem 272: 27401–27410, 1997.[Abstract/Free Full Text]
49. Chen S, Cao L, Intengan HD, Humphreys M, Gardner DG. Osmoregulation of endothelial nitric-oxide synthase gene expression in inner medullary collecting duct cells. Role in activation of the type A natriuretic peptide receptor. J Biol Chem 277: 32498–32504, 2002.[Abstract/Free Full Text]
50. Chen S, Gardner DG. Osmoregulation of natriuretic peptide receptor signaling in inner medullary collecting duct. A requirement for p38 MAPK. J Biol Chem 277: 6037–6043, 2002.[Abstract/Free Full Text]
51. Chung SS, Chung SK. Aldose reductase in diabetic microvascular complications. Curr Drug Targets 6: 475–486, 2005.[CrossRef][Web of Science][Medline]
52. Cohen DM. Urea-inducible Egr-1 transcription in renal inner medullary collecting duct (mIMCD3) cells is mediated by extracellular signal-regulated kinase activation. Proc Natl Acad Sci USA 93: 11242–11247, 1996.[Abstract/Free Full Text]
53. Cohen DM. SRC family kinases in cell volume regulation. Am J Physiol Cell Physiol 288: C483–C493, 2005.[Abstract/Free Full Text]
54. Cohen DM, Chin WW, Gullans SR. Hyperosmotic urea increases transcription and synthesis of Egr-1 in murine inner medullary collecting duct (mIMCD3) cells. J Biol Chem 269: 25865–25870, 1994.[Abstract/Free Full Text]
55. Cohen DM, Gullans SR. Urea selectively induces DNA synthesis in renal epithelial cells. Am J Physiol Renal Fluid Electrolyte Physiol 264: F601–F607, 1993.[Abstract/Free Full Text]
56. Cohen DM, Gullans SR. Urea induces Egr-1 and c-fos expression in renal epithelial cells. Am J Physiol Renal Fluid Electrolyte Physiol 264: F593–F600, 1993.[Abstract/Free Full Text]
57. Cohen DM, Gullans SR, Chin WW. Urea signaling in cultured murine inner medullary collecting duct (mIMCD3) cells involves protein kinase C, inositol 1,4,5-trisphosphate (IP₃), a putative receptor tyrosine kinase. J Clin Invest 97: 1884–1889, 1996.[Web of Science][Medline]
58. Cohen DM, Wasserman JC, Gullans SR. Immediate early gene and HSP70 expression in hyperosmotic stress in MDCK cells. Am J Physiol Cell Physiol 261: C594–C601, 1991.[Abstract/Free Full Text]
59. Colla E, Lee SD, Sheen MR, Woo SK, Kwon HM. TonEBP is inhibited by RNA helicase A via interaction involving the E'F loop. Biochem J 393: 411–419, 2005.[Web of Science]
60. Copp J, Wiley S, Ward MW, van der GP. Hypertonic shock inhibits growth factor receptor signaling, induces caspase-3 activation, causes reversible fragmentation of the mitochondrial network. Am J Physiol Cell Physiol 288: C403–C415, 2005.[Abstract/Free Full Text]
61. Cowley BD Jr, Muessel MJ, Douglass D, Wilkins W. In vivo and in vitro osmotic regulation of HSP-70 and prostaglandin synthase gene expression in kidney cells. Am J Physiol Renal Fluid Electrolyte Physiol 269: F854–F862, 1995.[Abstract/Free Full Text]
62. Cuenda A, Alonso G, Morrice N, Jones M, Meier R, Cohen P, Nebreda AR. Purification and cDNA cloning of SAPKK3, the major activator of RK/p38 in stress- and cytokine-stimulated monocytes and epithelial cells. EMBO J 15: 4156–4164, 1996.[Web of Science][Medline]
63. Cyert MS. Regulation of nuclear localization during signaling. J Biol Chem 276: 20805–20808, 2001.[Free Full Text]
64. D'Amours D, Jackson SP. The Mre11 complex: at the crossroads of DNA repair and checkpoint signalling. Nat Rev Mol Cell Biol 3: 317–327, 2002.[CrossRef][Web of Science][Medline]
65. Dahl SC, Handler JS, Kwon HM. Hypertonicity-induced phosphorylation and nuclear localization of the transcription factor TonEBP. Am J Physiol Cell Physiol 280: C248–C253, 2001.[Abstract/Free Full Text]
66. Dall'Asta V, Rossi PA, Bussolati O, Gazzola GC. Response of human fibroblasts to hypertonic stress. Cell shrinkage is counteracted by an enhanced active transport of neutral amino acids. J Biol Chem 269: 10485–10491, 1994.[Abstract/Free Full Text]
67. Dascalu A, Matithyou A, Oron Y, Korenstein R. A hyperosmotic stimulus elevates intracellular calcium and inhibits proliferation of a human keratinocyte cell line. J Invest Dermatol 115: 714–718, 2000.[CrossRef][Web of Science][Medline]
68. Di Ciano C, Nie Z, Szaszi K, Lewis A, Uruno T, Zhan X, Rotstein OD, Mak A, Kapus A. Osmotic stress-induced remodeling of the cortical cytoskeleton. Am J Physiol Cell Physiol 283: C850–C865, 2002.[Abstract/Free Full Text]
69. Di Ciano-Oliveira C, Sirokmany G, Szaszi K, Arthur WT, Masszi A, Peterson M, Rotstein OD, Kapus A. Hyperosmotic stress activates Rho: differential involvement in Rho kinase-dependent MLC phosphorylation and NKCC activation. Am J Physiol Cell Physiol 285: C555–C566, 2003.[Abstract/Free Full Text]
70. Dihazi H, Asif AR, Agarwal NK, Doncheva Y, Muller GA. Proteomic analysis of cellular response to osmotic stress in thick ascending limb of Henle's loop (TALH) cells. Mol Cell Proteomics 4: 1445–1458, 2005.[Abstract/Free Full Text]
71. Dmitrieva N, Kultz D, Michea L, Ferraris J, Burg M. Protection of renal inner medullary epithelial cells from apoptosis by hypertonic stress-induced p53 activation. J Biol Chem 275: 18243–18247, 2000.[Abstract/Free Full Text]
72. Dmitrieva N, Michea L, Burg MB. p53 tumor suppressor protein protects renal inner medullary cells from hypertonic stress by restricting DNA replication. Am J Physiol Renal Physiol 281: F522–F530, 2001.[Abstract/Free Full Text]
73. Dmitrieva NI, Bulavin DV, Burg MB. High NaCl causes Mre11 to leave the nucleus, disrupting DNA damage signaling and repair. Am J Physiol Renal Physiol 285: F266–F274, 2003.[Abstract/Free Full Text]
74. Dmitrieva NI, Bulavin DV, Fornace AJ Jr, Burg MB. Rapid activation of G2/M checkpoint after hypertonic stress in renal inner medullary epithelial (IME) cells is protective and requires p38 kinase. Proc Natl Acad Sci USA 99: 184–189, 2002.[Abstract/Free Full Text]
75. Dmitrieva NI, Cai Q, Burg MB. Cells adapted to high NaCl have many DNA breaks and impaired DNA repair both in cell culture and in vivo. Proc Natl Acad Sci USA 101: 2317–2322, 2004.[Abstract/Free Full Text]
76. Dmitrieva NI, Celeste A, Nussenzweig A, Burg MB. Ku86 preserves chromatin integrity in cells adapted to high NaCl. Proc Natl Acad Sci USA 102: 10730–10735, 2005.[Abstract/Free Full Text]
77. Eggermont J. Rho's role in cell volume: sensing, strutting, or signaling? Focus on "Hyperosmotic stress activates Rho: differential involvement in Rho kinase-dependent MLC phosphorylation and NKCC activation." Am J Physiol Cell Physiol 285: C509–C511, 2003.[Free Full Text]
78. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet 7: 606–619, 2006.[CrossRef][Web of Science][Medline]
79. Ericson AC, Spring KR. Volume regulation by Necturus gallbladder: apical Na⁺-H⁺ and Cl^–-HCO₃^– exchange. Am J Physiol Cell Physiol 243: C146–C150, 1982.[Abstract/Free Full Text]
80. Esensten JH, Tsytsykova AV, Lopez-Rodriguez C, Ligeiro FA, Rao A, Goldfeld AE. NFAT5 binds to the TNF promoter distinctly from NFATp, c, 3 and 4, activates TNF transcription during hypertonic stress alone. Nucleic Acids Res 33: 3845–3854, 2005.[Abstract/Free Full Text]
81. Feranchak AP, Kilic G, Wojtaszek PA, Qadri I, Fitz JG. Volume-sensitive tyrosine kinases regulate liver cell volume through effects on vesicular trafficking and membrane Na⁺ permeability. J Biol Chem 278: 44632–44638, 2003.[Abstract/Free Full Text]
82. Ferraris JD, Persaud P, Williams CK, Chen Y, Burg MB. cAMP-independent role of PKA in tonicity-induced transactivation of tonicity-responsive enhancer/osmotic response element-binding protein. Proc Natl Acad Sci USA 99: 16800–16805, 2002.[Abstract/Free Full Text]
83. Ferraris JD, Williams CK, Jung KY, Bedford JJ, Burg MB, Garcia-Perez A. ORE, a eukaryotic minimal essential osmotic response element. The aldose reductase gene in hyperosmotic stress. J Biol Chem 271: 18318–18321, 1996.[Abstract/Free Full Text]
84. Ferraris JD, Williams CK, Martin BM, Burg MB, Garcia-Perez A. Cloning, genomic organization, osmotic response of the aldose reductase gene. Proc Natl Acad Sci USA 91: 10742–10746, 1994.[Abstract/Free Full Text]
85. Ferraris JD, Williams CK, Ohtaka A, Garcia-Perez A. Functional consensus for mammalian osmotic response elements. Am J Physiol Cell Physiol 276: C667–C673, 1999.[Abstract/Free Full Text]
86. Ferraris JD, Williams CK, Persaud P, Zhang Z, Chen Y, Burg MB. Activity of the TonEBP/OREBP transactivation domain varies directly with extracellular NaCl concentration. Proc Natl Acad Sci USA 99: 739–744, 2002.[Abstract/Free Full Text]
87. Finkel T. Oxidant signals and oxidative stress. Curr Opin Cell Biol 15: 247–254, 2003.[CrossRef][Web of Science][Medline]
88. Friis MB, Friborg CR, Schneider L, Nielsen MB, Lambert IH, Christensen ST, Hoffmann EK. Cell shrinkage as a signal to apoptosis in NIH 3T3 fibroblasts. J Physiol 567: 427–443, 2005.[Abstract/Free Full Text]
89. Fritz A, Brayer KJ, McCormick N, Adams DG, Wadzinski BE, Vaillancourt RR. Phosphorylation of serine 526 is required for MEKK3 activity, association with 14–3-3 blocks dephosphorylation. J Biol Chem 281: 6236–6245, 2006.[Abstract/Free Full Text]
90. Furuta T, Takemura H, Liao ZY, Aune GJ, Redon C, Sedelnikova OA, Pilch DR, Rogakou EP, Celeste A, Chen HT, Nussenzweig A, Aladjem MI, Bonner WM, Pommier Y. Phosphorylation of histone H2AX and activation of Mre11, Rad50, Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes. J Biol Chem 278: 20303–20312, 2003.[Abstract/Free Full Text]
91. Gallazzini M, Ferraris JD, Kunin M, Morris RG, Burg MB. Neuropathy target esterase catalyzes osmoprotective renal synthesis of glycerophosphocholine in response to high NaCl. Proc Natl Acad Sci USA 103: 15260–15265, 2006.[Abstract/Free Full Text]
92. Gallazzini M, Karim Z, Bichara M. Regulation of ROMK (Kir 1.1) channel expression in kidney thick ascending limb by hypertonicity: role of TonEBP and MAPK pathways. Nephron Physiol 104: 126–135, 2006.[Medline]
93. Gallazzini M, ttmane-Elakeb A, Mount DB, Hebert SC, Bichara M. Regulation by glucocorticoids and osmolality of expression of ROMK (Kir 1.1), the apical K channel of thick ascending limb. Am J Physiol Renal Physiol 284: F977–F986, 2003.[Abstract/Free Full Text]
94. Galvez A, Morales MP, Eltit JM, Ocaranza P, Carrasco L, Campos X, Sapag-Hagar M, az-Araya G, Lavandero S. A rapid and strong apoptotic process is triggered by hyperosmotic stress in cultured rat cardiac myocytes. Cell Tissue Res 304: 279–285, 2001.[CrossRef][Web of Science][Medline]
95. Garcia-Perez A, Burg MB. Renal medullary organic osmolytes. Physiol Rev 71: 1081–1115, 1991.[Abstract/Free Full Text]
96. Garner MM, Burg MB. Macromolecular crowding and confinement in cells exposed to hypertonicity. Am J Physiol Cell Physiol 266: C877–C892, 1994.[Abstract/Free Full Text]
97. Garnovskaya MN, Mukhin YV, Vlasova TM, Raymond JR. Hypertonicity activates Na⁺/H⁺ exchange through Janus kinase 2 and calmodulin. J Biol Chem 278: 16908–16915, 2003.[Abstract/Free Full Text]
98. Gatsios P, Terstegen L, Schliess F, Haussinger D, Kerr IM, Heinrich PC, Graeve L. Activation of the Janus kinase/signal transducer and activator of transcription pathway by osmotic shock. J Biol Chem 273: 22962–22968, 1998.[Abstract/Free Full Text]
99. Gillin AG, Star RA, Sands JM. Osmolarity-stimulated urea transport in rat terminal IMCD: role of intracellular calcium. Am J Physiol Renal Fluid Electrolyte Physiol 265: F272–F277, 1993.[Abstract/Free Full Text]
100. Giono LE, Manfredi JJ. The p53 tumor suppressor participates in multiple cell cycle checkpoints. J Cell Physiol 209: 13–20, 2006.[CrossRef][Web of Science][Medline]
101. Go WY, Liu X, Roti MA, Liu F, Ho SN. NFAT5/TonEBP mutant mice define osmotic stress as a critical feature of the lymphoid microenvironment. Proc Natl Acad Sci USA 101: 10673–10678, 2004.[Abstract/Free Full Text]
102. Goedert M, Cuenda A, Craxton M, Jakes R, Cohen P. Activation of the novel stress-activated protein kinase SAPK4 by cytokines and cellular stresses is mediated by SKK3 (MKK6): comparison of its substrate specificity with that of other SAP kinases. EMBO J 16: 3563–3571, 1997.[CrossRef][Web of Science][Medline]
103. Good DW, Di Mari JF, Watts BA III. Hyposmolality stimulates Na⁺/H⁺ exchange and HCO₃^– absorption in thick ascending limb via PI 3-kinase. Am J Physiol Cell Physiol 279: C1443–C1454, 2000.[Abstract/Free Full Text]
104. Greenberg MM. In vitro and in vivo effects of oxidative damage to deoxyguanosine. Biochem Soc Trans 32: 46–50, 2004.[CrossRef][Web of Science][Medline]
105. Grinstein S, Rothstein A, Cohen S. Mechanism of osmotic activation of Na⁺/H⁺ exchange in rat thymic lymphocytes. J Gen Physiol 85: 765–787, 1985.[Abstract/Free Full Text]
106. Grunewald RW, Wagner M, Schubert I, Franz HE, Muller GA, Steffgen J. Rat renal expression of mRNA coding for aldose reductase and sorbitol dehydrogenase and its osmotic regulation in inner medullary collecting duct cells. Cell Physiol Biochem 8: 293–303, 1998.[CrossRef][Web of Science][Medline]
107. Gual P, Gonzalez T, Gremeaux T, Barres R, Le Marchand-Brustel Y, Tanti JF. Hyperosmotic stress inhibits insulin receptor substrate-1 function by distinct mechanisms in 3T3–L1 adipocytes. J Biol Chem 278: 26550–26557, 2003.[Abstract/Free Full Text]
108. Gual P, Shigematsu S, Kanzaki M, Gremeaux T, Gonzalez T, Pessin JE, Le Marchand-Brustel Y, Tanti JF. A Crk-II/TC10 signaling pathway is required for osmotic shock-stimulated glucose transport. J Biol Chem 277: 43980–43986, 2002.[Abstract/Free Full Text]
109. Hager K, Hazama A, Kwon HM, Loo DD, Handler JS, Wright EM. Kinetics and specificity of the renal Na⁺/myo-inositol cotransporter expressed in Xenopus oocytes. J Membr Biol 143: 103–113, 1995.[Web of Science][Medline]
110. Hallows KR, Law FY, Packman CH, Knauf PA. Changes in cytoskeletal actin content, F-actin distribution, surface morphology during HL-60 cell volume regulation. J Cell Physiol 167: 60–71, 1996.[CrossRef][Web of Science][Medline]
111. Han J, Lee JD, Bibbs L, Ulevitch RJ. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265: 808–811, 1994.[Abstract/Free Full Text]
112. Hao CM, Redha R, Morrow J, Breyer MD. Peroxisome proliferator-activated receptor delta activation promotes cell survival following hypertonic stress. J Biol Chem 277: 21341–21345, 2002.[Abstract/Free Full Text]
113. Hasler U, Jeon US, Kim JA, Mordasini D, Kwon HM, Feraille E, Martin PY. Tonicity-responsive enhancer binding protein is an essential regulator of aquaporin-2 expression in renal collecting duct principal cells. J Am Soc Nephrol 17: 1521–1531, 2006.[Abstract/Free Full Text]
114. Hasler U, Vinciguerra M, Vandewalle A, Martin PY, Feraille E. Dual effects of hypertonicity on aquaporin-2 expression in cultured renal collecting duct principal cells. J Am Soc Nephrol 16: 1571–1582, 2005.[Abstract/Free Full Text]
115. Head MW, Corbin E, Goldman JE. Coordinate and independent regulation of alpha B-crystallin and hsp27 expression in response to physiological stress. J Cell Physiol 159: 41–50, 1994.[CrossRef][Web of Science][Medline]
116. Herrlich A, Leitch V, King LS. Role of proneuregulin 1 cleavage and human epidermal growth factor receptor activation in hypertonic aquaporin induction. Proc Natl Acad Sci USA 101: 15799–15804, 2004.[Abstract/Free Full Text]
117. Hoffert JD, Leitch V, Agre P, King LS. Hypertonic induction of aquaporin-5 expression through an ERK-dependent pathway. J Biol Chem 275: 9070–9077, 2000.[Abstract/Free Full Text]
118. Huang Z, Tunnacliffe A. Gene induction by desiccation stress in human cell cultures. FEBS Lett 579: 4973–4977, 2005.[CrossRef][Web of Science][Medline]
119. Huangfu WC, Omori E, Akira S, Matsumoto K, Ninomiya-Tsuji J. Osmotic stress activates the TAK1-JNK pathway while blocking TAK1-mediated NF-kappa B activation: TAO2 regulates TAK1 pathways. J Biol Chem 281: 28802–28810, 2006.[Abstract/Free Full Text]
120. Hwang DY, Ismail-Beigi F. Stimulation of GLUT-1 glucose transporter expression in response to hyperosmolarity. Am J Physiol Cell Physiol 281: C1365–C1372, 2001.[Abstract/Free Full Text]
121. Hwang YC, Kaneko M, Bakr S, Liao H, Lu Y, Lewis ER, Yan S, Ii S, Itakura M, Rui L, Skopicki H, Homma S, Schmidt AM, Oates PJ, Szabolcs M, Ramasamy R. Central role for aldose reductase pathway in myocardial ischemic injury. FASEB J 18: 1192–1199, 2004.[Abstract/Free Full Text]
122. Iliakis G, Wang Y, Guan J, Wang H. DNA damage checkpoint control in cells exposed to ionizing radiation. Oncogene 22: 5834–5847, 2003.[CrossRef][Web of Science][Medline]
123. Irarrazabal CE, Burg MB, Ward SG, Ferraris JD. Phosphatidylinositol 3-kinase mediates activation of ATM by high NaCl and by ionizing radiation: role in osmoprotective transcriptional regulation. Proc Natl Acad Sci USA 103: 8882–8887, 2006.[Abstract/Free Full Text]
124. Irarrazabal CE, Liu JC, Burg MB, Ferraris JD. ATM, a DNA damage-inducible kinase, contributes to activation by high NaCl of the transcription factor TonEBP/OREBP. Proc Natl Acad Sci USA 101: 8809–8814, 2004.[Abstract/Free Full Text]
125. Ito H, Okamoto K, Nakayama H, Isobe T, Kato K. Phosphorylation of alphaB-crystallin in response to various types of stress. J Biol Chem 272: 29934–29941, 1997.[Abstract/Free Full Text]
126. Ito T, Fujio Y, Hirata M, Takatani T, Matsuda T, Muraoka S, Takahashi K, Azuma J. Expression of taurine transporter is regulated through the TonE (tonicity-responsive element)/TonEBP (TonE-binding protein) pathway and contributes to cytoprotection in HepG2 cells. Biochem J 382: 177–182, 2004.[CrossRef][Web of Science][Medline]
127. Itoh T, Yamauchi A, Miyai A, Yokoyama K, Kamada T, Ueda N, Fujiwara Y. Mitogen-activated protein kinase and its activator are regulated by hypertonic stress in Madin-Darby canine kidney cells. J Clin Invest 93: 2387–2392, 1994.[Web of Science][Medline]
128. Janez A, Worrall DS, Imamura T, Sharma PM, Olefsky JM. The osmotic shock-induced glucose transport pathway in 3T3–L1 adipocytes is mediated by gab-1 and requires Gab-1-associated phosphatidylinositol 3-kinase activity for full activation. J Biol Chem 275: 26870–26876, 2000.[Abstract/Free Full Text]
129. Jenq W, Mathieson IM, Ihara W, Ramirez G. Aquaporin-1: an osmoinducible water channel in cultured mIMCD-3 cells. Biochem Biophys Res Commun 245: 804–809, 1998.[CrossRef][Web of Science][Medline]
130. Jiang HY, Wek SA, McGrath BC, Lu D, Hai T, Harding HP, Wang X, Ron D, Cavener DR, Wek RC. Activating transcription factor 3 is integral to the eukaryotic initiation factor 2 kinase stress response. Mol Cell Biol 24: 1365–1377, 2004.[Abstract/Free Full Text]
131. Jiang Y, Chen C, Li Z, Guo W, Gegner JA, Lin S, Han J. Characterization of the structure and function of a new mitogen-activated protein kinase (p38beta). J Biol Chem 271: 17920–17926, 1996.[Abstract/Free Full Text]
132. Jin Z, El-Deiry WS. Overview of cell death signaling pathways. Cancer Biol Ther 4: 139–163, 2005.[Web of Science][Medline]
133. Kaiserova K, Srivastava S, Hoetker JD, Awe SO, Tang XL, Cai J, Bhatnagar A. Redox activation of aldose reductase in the ischemic heart. J Biol Chem 281: 15110–15120, 2006.[Abstract/Free Full Text]
134. Kang YJ, Seit-Nebi A, Davis RJ, Han J. Multiple activation mechanisms of p38alpha mitogen-activated protein kinase. J Biol Chem 281: 26225–26234, 2006.[Abstract/Free Full Text]
135. Kapus A, Di CC, Sun J, Zhan X, Kim L, Wong TW, Rotstein OD. Cell volume-dependent phosphorylation of proteins of the cortical cytoskeleton and cell-cell contact sites. The role of Fyn and FER kinases. J Biol Chem 275: 32289–32298, 2000.[Abstract/Free Full Text]
136. Kapus A, Szaszi K, Sun J, Rizoli S, Rotstein OD. Cell shrinkage regulates Src kinases and induces tyrosine phosphorylation of cortactin, independent of the osmotic regulation of Na⁺/H⁺ exchangers. J Biol Chem 274: 8093–8102, 1999.[Abstract/Free Full Text]
137. Kempson SA, Beck JA, Lammers PE, Gens JS, Montrose MH. Membrane insertion of betaine/GABA transporter during hypertonic stress correlates with nuclear accumulation of TonEBP. Biochim Biophys Acta 1712: 71–80, 2005.[Medline]
138. Kempson SA, Edwards JM, Sturek M. Inhibition of the renal betaine transporter by calcium ions. Am J Physiol Renal Physiol 291: F305–F313, 2006.[Abstract/Free Full Text]
139. Kempson SA, Montrose MH. Osmotic regulation of renal betaine transport: transcription and beyond. Pflügers Arch 449: 227–234, 2004.[Web of Science][Medline]
140. Kim DK, Jung KY. Caffeine causes glycerophosphorylcholine accumulation through ryanodine-inhibitable increase of cellular calcium and activation of phospholipase A₂ in cultured MDCK cells. Exp Mol Med 30: 151–158, 1998.[Web of Science][Medline]
141. Klein JD, O'Neill WC. Volume-sensitive myosin phosphorylation in vascular endothelial cells: correlation with Na-K-2Cl cotransport. Am J Physiol Cell Physiol 269: C1524–C1531, 1995.[Abstract/Free Full Text]
142. Ko BC, Lam AK, Kapus A, Fan L, Chung SK, Chung SS. Fyn and p38 signaling are both required for maximal hypertonic activation of the OREBP/TonEBP. J Biol Chem 277: 46085–46092, 2002.[Abstract/Free Full Text]
143. Ko BC, Turck CW, Lee KW, Yang Y, Chung SS. Purification, identification, characterization of an osmotic response element binding protein. Biochem Biophys Res Commun 270: 52–61, 2000.[CrossRef][Web of Science][Medline]
144. Ko BCB, Ruepp B, Bohren KM, Gabbay KH, Chung SS. Identification and characterization of multiple osmotic response sequences in the human aldose reductase gene. J Biol Chem 272: 16431–16437, 1997.[Abstract/Free Full Text]
145. Koch JP, Korbmacher C. Mechanism of shrinkage activation of nonselective cation channels in M-1 mouse cortical collecting duct cells. J Membr Biol 177: 231–242, 2000.[CrossRef][Web of Science][Medline]
146. Kojima R, Randall JD, Ito E, Manshio H, Suzuki Y, Gullans SR. Regulation of expression of the stress response gene, Osp94: identification of the tonicity response element and intracellular signalling pathways. Biochem J 380: 783–794, 2004.[CrossRef][Web of Science][Medline]
147. Konishi H, Matsuzaki H, Tanaka M, Ono Y, Tokunaga C, Kuroda S, Kikkawa U. Activation of RAC-protein kinase by heat shock and hyperosmolarity stress through a pathway independent of phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA 93: 7639–7643, 1996.[Abstract/Free Full Text]
148. Kozak M. Leader length and secondary structure modulate mRNA function under conditions of stress. Mol Cell Biol 8: 2737–2744, 1988.[Abstract/Free Full Text]
149. Kramer HJ, Hashemi T, Backer A, Bokemeyer D. Hyperosmolality induced by betaine or urea stimulates endothelin synthesis by differential activation of ERK and p38 MAP kinase in MDCK cells. Kidney Blood Press Res 25: 65–70, 2002.[CrossRef][Web of Science][Medline]
150. Krarup T, Jakobsen LD, Jensen BS, Hoffmann EK. Na⁺-K⁺-2Cl^– cotransport in Ehrlich cells: regulation by protein phosphatases and kinases. Am J Physiol Cell Physiol 275: C239–C250, 1998.[Abstract/Free Full Text]
151. Krump E, Nikitas K, Grinstein S. Induction of tyrosine phosphorylation and Na⁺/H⁺ exchanger activation during shrinkage of human neutrophils. J Biol Chem 272: 17303–17311, 1997.[Abstract/Free Full Text]
152. Kultz D. Osmotic regulation of DNA activity and the cell cycle. In: Environmental Stresses and Gene Responses, edited by Storey KB and Storey J. Amsterdam: Elsevier Science, 2000, p. 157–179.
153. Kultz D, Chakravarty D. Hyperosmolality in the form of elevated NaCl but not urea causes DNA damage in murine kidney cells. Proc Natl Acad Sci USA 98: 1999–2004, 2001.[Abstract/Free Full Text]
154. Kultz D, Garcia-Perez A, Ferraris JD, Burg MB. Distinct regulation of osmoprotective genes in yeast and mammals. Aldose reductase osmotic response element is induced independent of p38 and stress-activated protein kinase/Jun N-terminal kinase in rabbit kidney cells. J Biol Chem 272: 13165–13170, 1997.[Abstract/Free Full Text]
155. Kultz D, Madhany S, Burg MB. Hyperosmolality causes growth arrest of murine kidney cells. Induction of GADD45 and GADD153 by osmosensing via stress-activated protein kinase 2. J Biol Chem 273: 13645–13651, 1998.[Abstract/Free Full Text]
156. Kwon ED, Jung KY, Edsall LC, Kim HY, Garcia-Perez A, Burg MB. Osmotic regulation of synthesis of glycerophosphocholine from phosphatidylcholine in MDCK cells. Am J Physiol Cell Physiol 268: C402–C412, 1995.[Abstract/Free Full Text]
157. Kwon ED, Zablocki K, Jung KY, Peters EM, Garcia-Perez A, Burg MB. Osmoregulation of GPC:choline phosphodiesterase in MDCK cells: different effects of urea and NaCl. Am J Physiol Cell Physiol 269: C35–C41, 1995.[Abstract/Free Full Text]
158. Kwon ED, Zablocki K, Peters EM, Jung KY, Garcia-Perez A, Burg MB. Betaine and inositol reduce MDCK cell glycerophosphocholine by stimulating its degradation. Am J Physiol Cell Physiol 270: C200–C207, 1996.[Abstract/Free Full Text]
159. Kwon HM, Itoh T, Rim JS, Handler JS. The MAP kinase cascade is not essential for transcriptional stimulation of osmolyte transporter genes. Biochem Biophys Res Commun 213: 975–979, 1995.[CrossRef][Web of Science][Medline]
160. Kwon HM, Yamauchi A, Uchida S, Preston AS, Garcia-Perez A, Burg MB, Handler JS. Cloning of the cDNA for a Na⁺/myo-inositol cotransporter, a hypertonicity stress protein. J Biol Chem 267: 6297–6301, 1992.[Abstract/Free Full Text]
161. Lam AK, Ko BC, Tam S, Morris R, Yang JY, Chung SK, Chung SS. Osmotic response element-binding protein (OREBP) is an essential regulator of the urine concentrating mechanism. J Biol Chem 279: 48048–48054, 2004.[Abstract/Free Full Text]
162. Lang KS, Fillon S, Schneider D, Rammensee HG, Lang F. Stimulation of TNF alpha expression by hyperosmotic stress. Pflügers Arch 443: 798–803, 2002.[CrossRef][Web of Science][Medline]
163. Larsen AK, Jensen BS, Hoffmann EK. Activation of protein kinase C during cell volume regulation in Ehrlich mouse ascites tumor cells. Biochim Biophys Acta 1222: 477–482, 1994.[Medline]
164. Lee JS, Lee JJ, Seo JS. HSP70 deficiency results in activation of c-Jun N-terminal kinase, extracellular signal-regulated kinase, caspase-3 in hyperosmolarity-induced apoptosis. J Biol Chem 280: 6634–6641, 2005.[Abstract/Free Full Text]
165. Lee SD, Colla E, Sheen MR, Na KY, Kwon HM. Multiple domains of TonEBP cooperate to stimulate transcription in response to hypertonicity. J Biol Chem 278: 47571–47577, 2003.[Abstract/Free Full Text]
166. Lee SD, Woo SK, Kwon HM. Dimerization is required for phosphorylation and DNA binding of TonEBP/NFAT5. Biochem Biophys Res Commun 294: 968–975, 2002.[CrossRef][Web of Science][Medline]
167. Leroy C, Basset G, Gruel G, Ripoche P, Trinh-Trang-Tan MM, Rousselet G. Hyperosmotic NaCl and urea synergistically regulate the expression of the UT-A2 urea transporter in vitro and in vivo. Biochem Biophys Res Commun 271: 368–373, 2000.[CrossRef][Web of Science][Medline]
168. Leroy C, Colmont C, Pisam M, Rousselet G. Different responses to acute or progressive osmolarity increases in the mIMCD3 cell line. Eur J Cell Biol 79: 936–942, 2000.[CrossRef][Web of Science][Medline]
169. Levine RL. Carbonyl modified proteins in cellular regulation, aging, disease. Free Radical Biol Med 32: 790–796, 2002.[CrossRef][Web of Science][Medline]
170. Levine RL, Wehr N, Williams JA, Stadtman ER, Shacter E. Determination of carbonyl groups in oxidized proteins. Methods Mol Biol 99: 15–24, 2000.[Medline]
171. Lewis A, Di CC, Rotstein OD, Kapus A. Osmotic stress activates Rac and Cdc42 in neutrophils: role in hypertonicity-induced actin polymerization. Am J Physiol Cell Physiol 282: C271–C279, 2002.[Abstract/Free Full Text]
172. Lin KW, Nam SY, Toh WH, Dulloo I, Sabapathy K. Multiple stress signals induce p73beta accumulation. Neoplasia 6: 546–557, 2004.[CrossRef][Web of Science][Medline]
173. Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini-Rivera S, DeMayo F, Bradley A, Donehower LA, Elledge SJ. Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev 14: 1448–1459, 2000.[Abstract/Free Full Text]
174. Logan JL. Studies on renal growth regulation by urea and ammonia in normal rat kidney cells. Nephron 71: 433–441, 1995.[Web of Science][Medline]
175. Lohr JW, Grantham JJ. Isovolumetric regulation of isolated S2 proximal tubules in anisotonic media. J Clin Invest 78: 1165–1172, 1986.[Web of Science][Medline]
176. Lopez-Rodriguez C, Antos CL, Shelton JM, Richardson JA, Lin F, Novobrantseva TI, Bronson RT, Igarashi P, Rao A, Olson EN. Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression. Proc Natl Acad Sci USA 101: 2392–2397, 2004.[Abstract/Free Full Text]
177. Lopez-Rodriguez C, Aramburu J, Jin L, Rakeman AS, Michino M, Rao A. Bridging the NFAT and NF-kappaB families: NFAT5 dimerization regulates cytokine gene transcription in response to osmotic stress. Immunity 15: 47–58, 2001.[CrossRef][Web of Science][Medline]
178. Lopez-Rodriguez C, Aramburu J, Rakeman AS, Copeland NG, Gilbert DJ, Thomas S, Disteche C, Jenkins NA, Rao A. NF-AT5: the NF-AT family of transcription factors expands in a new direction. Cold Spring Harb Symp Quant Biol 64: 517–526, 1999.[CrossRef][Web of Science][Medline]
179. Lopez-Rodriguez C, Aramburu J, Rakeman AS, Rao A. NFAT5, a constitutively nuclear NFAT protein that does not cooperate with Fos and Jun. Proc Natl Acad Sci USA 96: 7214–7219, 1999.[Abstract/Free Full Text]
180. Low SY, Taylor PM. Integrin and cytoskeletal involvement in signalling cell volume changes to glutamine transport in rat skeletal muscle. J Physiol 512: 481–485, 1998.[Abstract/Free Full Text]
181. Lu L, Han AP, Chen JJ. Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses. Mol Cell Biol 21: 7971–7980, 2001.[Abstract/Free Full Text]
182. Lunn JA, Rozengurt E. Hyperosmotic stress induces rapid focal adhesion kinase phosphorylation at tyrosines 397 and 577. Role of Src family kinases and Rho family GTPases. J Biol Chem 279: 45266–45278, 2004.[Abstract/Free Full Text]
183. Luo G, Yao MS, Bender CF, Mills M, Bladl AR, Bradley A, Petrini JH. Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, sensitivity to ionizing radiation. Proc Natl Acad Sci USA 96: 7376–7381, 1999.[Abstract/Free Full Text]
184. Maeno E, Ishizaki Y, Kanaseki T, Hazama A, Okada Y. Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proc Natl Acad Sci USA 97: 9487–9492, 2000.[Abstract/Free Full Text]
185. Maeno E, Takahashi N, Okada Y. Dysfunction of regulatory volume increase is a key component of apoptosis. FEBS Lett 580: 6513–6517, 2006.[CrossRef][Web of Science][Medline]
186. Mak SK, Kultz D. Gadd45 proteins induce G₂/M arrest and modulate apoptosis in kidney cells exposed to hyperosmotic stress. J Biol Chem 279: 39075–39084, 2004.[Abstract/Free Full Text]
187. Mallee JJ, Atta MG, Lorica V, Rim JS, Kwon HM, Lucente AD, Wang Y, Berry GT. The structural organization of the human Na⁺/myo-inositol cotransporter (SLC5A3) gene and characterization of the promoter. Genomics 46: 459–465, 1997.[CrossRef][Web of Science][Medline]
188. Maric C, Casley D, Harris P, Alcorn D. Angiotensin II binding to renomedullary interstitial cells is regulated by osmolality. J Am Soc Nephrol 12: 450–455, 2001.[Abstract/Free Full Text]
189. Marsh DJ, Azen SP. Mechanism of NaCl reabsorption by hamster thin ascending limbs of Henle's loop. Am J Physiol 228: 71–79, 1975.[Abstract/Free Full Text]
190. Matsuda S, Kawasaki H, Moriguchi T, Gotoh Y, Nishida E. Activation of protein kinase cascades by osmotic shock. J Biol Chem 270: 12781–12786, 1995.[Abstract/Free Full Text]
191. Matsuzaki T, Suzuki T, Takata K. Hypertonicity-induced expression of aquaporin 3 in MDCK cells. Am J Physiol Cell Physiol 281: C55–C63, 2001.[Abstract/Free Full Text]
192. McReynolds MR, Taylor-Garcia KM, Greer KA, Hoying JB, Brooks HL. Renal medullary gene expression in aquaporin-1 null mice. Am J Physiol Renal Physiol 288: F315–F321, 2005.[Abstract/Free Full Text]
193. Meek K, Gupta S, Ramsden DA, Lees-Miller SP. The DNA-dependent protein kinase: the director at the end. Immunol Rev 200: 132–41.: 132–141, 2004.[CrossRef][Web of Science][Medline]
194. Meier R, Rouse J, Cuenda A, Nebreda AR, Cohen P. Cellular stresses and cytokines activate multiple mitogen-activated-protein kinase kinase homologues in PC12 and KB cells. Eur J Biochem 236: 796–805, 1996.[Web of Science][Medline]
195. Meier R, Thelen M, Hemmings BA. Inactivation and dephosphorylation of protein kinase Balpha (PKBalpha) promoted by hyperosmotic stress. EMBO J 17: 7294–7303, 1998.[CrossRef][Web of Science][Medline]
196. Miah SM, Sada K, Tuazon PT, Ling J, Maeno K, Kyo S, Qu X, Tohyama Y, Traugh JA, Yamamura H. Activation of Syk protein tyrosine kinase in response to osmotic stress requires interaction with p21-activated protein kinase Pak2/gamma-PAK. Mol Cell Biol 24: 71–83, 2004.[Abstract/Free Full Text]
197. Michea L, Combs C, Peters EM, Dmitrieva N, Andrews PM, Burg MB. Mitochondrial dysfunction is an early event in high NaCl-induced apoptosis of mIMCD-3 cells. Am J Physiol Renal Physiol 282: F981–F990, 2002.[Abstract/Free Full Text]
198. Michea L, Ferguson DR, Peters EM, Andrews PM, Kirby MR, Burg MB. Cell cycle delay and apoptosis are induced by high salt and urea in renal medullary cells. Am J Physiol Renal Physiol 278: F209–F218, 2000.[Abstract/Free Full Text]
199. Mitruka BM, Rawnsley HM. Clinical Biochemical and Hematological Reference Values in Normal Experimental Animals. New York: Masson, 1977.
200. Miyakawa H, Rim JS, Handler JS, Kwon HM. Identification of the second tonicity-responsive enhancer for the betaine transporter (BGT1) gene. Biochim Biophys Acta 1446: 359–364, 1999.[Medline]
201. Miyakawa H, Woo SK, Dahl SC, Handler JS, Kwon HM. Tonicity-responsive enhancer binding protein, a Rel-like protein that stimulates transcription in response to hypertonicity. Proc Natl Acad Sci USA 96: 2538–2542, 1999.[Abstract/Free Full Text]
202. Moeckel GW, Zhang L, Chen X, Rossini M, Zent R, Pozzi A. Role of integrin alpha1beta1 in the regulation of renal medullary osmolyte concentration. Am J Physiol Renal Physiol 290: F223–F231, 2006.[Abstract/Free Full Text]
203. Moeslinger T, Friedl R, Volf I, Brunner M, Baran H, Koller E, Spieckermann PG. Urea induces macrophage proliferation by inhibition of inducible nitric oxide synthesis. Kidney Int 56: 581–588, 1999.[CrossRef][Web of Science][Medline]
204. Moriyama T, Garcia-Perez A, Burg MB. Factors affecting the ratio of different organic osmolytes in renal medullary cells. Am J Physiol Renal Fluid Electrolyte Physiol 259: F847–F858, 1990.[Abstract/Free Full Text]
205. Moriyama T, Garcia-Perez A, Olson AD, Burg MB. Intracellular betaine substitutes for sorbitol in protecting renal medullary cells from hypertonicity. Am J Physiol Renal Fluid Electrolyte Physiol 260: F494–F497, 1991.[Abstract/Free Full Text]
206. Morse D, Choi AM. Heme oxygenase-1: the "emerging molecule" has arrived. Am J Respir Cell Mol Biol 27: 8–16, 2002.[Abstract/Free Full Text]
207. Mountian I, Waelkens E, Missiaen L, Van Driessche W. Changes in actin cytoskeleton during volume regulation in C6 glial cells. Eur J Cell Biol 77: 196–204, 1998.[Web of Science][Medline]
208. Mountian I, Van Driessche W. Isovolumetric regulation of C6 rat glioma cells in hyperosmotic media. Am J Physiol Cell Physiol 272: C318–C323, 1997.[Abstract/Free Full Text]
209. Munoz A, Alonso MA, Carrasco L. Synthesis of heat-shock proteins in HeLa cells: inhibition by virus infection. Virology 137: 150–159, 1984.[CrossRef][Web of Science][Medline]
210. Nadkarni V, Gabbay KH, Bohren KM, Sheikh-Hamad D. Osmotic response element enhancer activity. Regulation through p38 kinase and mitogen-activated extracellular signal-regulated kinase kinase. J Biol Chem 274: 20185–20190, 1999.[Abstract/Free Full Text]
211. Nahm O, Woo SK, Handler JS, Kwon HM. Involvement of multiple kinase pathways in stimulation of gene transcription by hypertonicity. Am J Physiol Cell Physiol 282: C49–C58, 2002.[Abstract/Free Full Text]
212. Nakanishi T, Balaban RS, Burg MB. Survey of osmolytes in renal cell lines. Am J Physiol Cell Physiol 255: C181–C191, 1988.[Abstract/Free Full Text]
213. Nakanishi T, Turner RJ, Burg MB. Osmoregulatory changes in myo-inositol transport by renal cells. Proc Natl Acad Sci USA 86: 6002–6006, 1989.[Abstract/Free Full Text]
214. Nakanishi T, Turner RJ, Burg MB. Osmoregulation of betaine transport in mammalian renal medullary cells. Am J Physiol Renal Fluid Electrolyte Physiol 258: F1061–F1067, 1990.[Abstract/Free Full Text]
215. Nakanishi T, Uyama O, Sugita M. Osmotically regulated taurine content in rat renal inner medulla. Am J Physiol Renal Fluid Electrolyte Physiol 261: F957–F962, 1991.[Abstract/Free Full Text]
216. Nakayama Y, Peng T, Sands JM, Bagnasco SM. The TonE/TonEBP pathway mediates tonicity-responsive regulation of UT-A urea transporter expression. J Biol Chem 275: 38275–38280, 2000.[Abstract/Free Full Text]
217. Nemeth ZH, Deitch EA, Szabo C, Hasko G. Hyperosmotic stress induces nuclear factor-kappaB activation and interleukin-8 production in human intestinal epithelial cells. Am J Pathol 161: 987–996, 2002.[Abstract/Free Full Text]
218. Neuhofer W, Lugmayr K, Fraek ML, Beck FX. Regulated overexpression of heat shock protein 72 protects Madin-Darby canine kidney cells from the detrimental effects of high urea concentrations. J Am Soc Nephrol 12: 2565–2571, 2001.[Abstract/Free Full Text]
219. Neuhofer W, Muller E, Burger-Kentischer A, Fraek ML, Thurau K, Beck F. Pretreatment with hypertonic NaCl protects MDCK cells against high urea concentrations. Pflügers Arch 435: 407–414, 1998.[CrossRef][Web of Science][Medline]
220. Neuhofer W, Woo SK, Na KY, Grunbein R, Park WK, Nahm O, Beck FX, Kwon HM. Regulation of TonEBP transcriptional activator in MDCK cells following changes in ambient tonicity. Am J Physiol Cell Physiol 283: C1604–C1611, 2002.[Abstract/Free Full Text]
221. Niswander JM, Dokas LA. Phosphorylation of HSP27 and synthesis of 14–3-3epsilon are parallel responses to hyperosmotic stress in the hippocampus. Brain Res 1116: 19–30, 2006.[CrossRef][Web of Science][Medline]
222. Nussenzweig A, Sokol K, Burgman P, Li L, Li GC. Hypersensitivity of Ku80-deficient cell lines and mice to DNA damage: the effects of ionizing radiation on growth, survival, development. Proc Natl Acad Sci USA 94: 13588–13593, 1997.[Abstract/Free Full Text]
223. Ohtaka A, Muto S, Nemoto J, Kawakami K, Nagano K, Asano Y. Hyperosmolality stimulates Na-K-ATPase gene expression in inner medullary collecting duct cells. Am J Physiol Renal Fluid Electrolyte Physiol 270: F728–F738, 1996.[Abstract/Free Full Text]
224. Okada Y, Maeno E, Shimizu T, Dezaki K, Wang J, Morishima S. Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD). J Physiol 532: 3–16, 2001.[Abstract/Free Full Text]
225. Ouwens DM, Gomes de Mesquita DS, Dekker J, Maassen JA. Hyperosmotic stress activates the insulin receptor in CHO cells. Biochim Biophys Acta 1540: 97–106, 2001.[Medline]
226. Padda R, Wamsley-Davis AM, Gustin MC, Ross R, Yu C, Sheikh-Hamad D. MEKK3-mediated signaling to p38 kinase and TonE in hypertonically stressed kidney cells. Am J Physiol Renal Physiol 291: F874–F881, 2006.[Abstract/Free Full Text]
227. Pandey P, Avraham S, Kumar S, Nakazawa A, Place A, Ghanem L, Rana A, Kumar V, Majumder PK, Avraham H, Davis RJ, Kharbanda S. Activation of p38 mitogen-activated protein kinase by PYK2/related adhesion focal tyrosine kinase-dependent mechanism. J Biol Chem 274: 10140–10144, 1999.[Abstract/Free Full Text]
228. Parrott LA, Templeton DJ. Osmotic stress inhibits p70/85 S6 kinase through activation of a protein phosphatase. J Biol Chem 274: 24731–24736, 1999.[Abstract/Free Full Text]
229. Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 10: 886–895, 2000.[CrossRef][Web of Science][Medline]
230. Pedersen SF, Hoffmann EK. Possible interrelationship between changes in F-actin and myosin II, protein phosphorylation, cell volume regulation in Ehrlich ascites tumor cells. Exp Cell Res 277: 57–73, 2002.[CrossRef][Web of Science][Medline]
231. Pedersen SF, Hoffmann EK, Mills JW. The cytoskeleton and cell volume regulation. Comp Biochem Physiol A Mol Integr Physiol 130: 385–399, 2001.[CrossRef][Medline]
232. Pederson T, Robbins E. RNA synthesis in HeLa cells. Pattern in hypertonic medium and its similarity to synthesis during G₂-prophase. J Cell Biol 47: 734–744, 1970.[Abstract/Free Full Text]
233. Perez-Pinera P, Menendez-Gonzalez M, Valle MD, Vega JA. Hypertonicity activates GSK3beta in tumor cells. Mol Cell Biochem. In press.
234. Pesesse X, Choi K, Zhang T, Shears SB. Signaling by higher inositol polyphosphates. Synthesis of bisdiphosphoinositol tetrakisphosphate ("InsP8") is selectively activated by hyperosmotic stress. J Biol Chem 279: 43378–43381, 2004.[Abstract/Free Full Text]
235. Petronini PG, Tramacere M, Kay JE, Borghetti AF. Adaptive response of cultured fibroblasts to hyperosmolarity. Exp Cell Res 165: 180–190, 1986.[CrossRef][Web of Science][Medline]
236. Prabhu KS, Arner RJ, Vunta H, Reddy CC. Up-regulation of human myo-inositol oxygenase by hyperosmotic stress in renal proximal tubular epithelial cells. J Biol Chem 280: 19895–19901, 2005.[Abstract/Free Full Text]
237. Qin S, Minami Y, Hibi M, Kurosaki T, Yamamura H. Syk-dependent and -independent signaling cascades in B cells elicited by osmotic and oxidative stress. J Biol Chem 272: 2098–2103, 1997.[Abstract/Free Full Text]
238. Ramadoss P, Perdew GH. The transactivation domain of the Ah receptor is a key determinant of cellular localization and ligand-independent nucleocytoplasmic shuttling properties. Biochemistry 44: 11148–11159, 2005.[CrossRef][Medline]
239. Rao R, Hao CM, Breyer MD. Hypertonic stress activates glycogen synthase kinase 3beta-mediated apoptosis of renal medullary interstitial cells, suppressing an NFkappaB-driven cyclooxygenase-2-dependent survival pathway. J Biol Chem 279: 3949–3955, 2004.[Abstract/Free Full Text]
240. Rauchman MI, Nigam SK, Delpire E, Gullans SR. An osmotically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse. Am J Physiol Renal Fluid Electrolyte Physiol 265: F416–F424, 1993.[Abstract/Free Full Text]
241. Rauchman MI, Pullman J, Gullans SR. Induction of molecular chaperones by hyperosmotic stress in mouse inner medullary collecting duct cells. Am J Physiol Renal Physiol 273: F9–F17, 1997.[Abstract/Free Full Text]
242. Reinehr R, Becker S, Braun J, Eberle A, Grether-Beck S, Haussinger D. Endosomal acidification and activation of NADPH oxidase isoforms are upstream events in hyperosmolarity-induced hepatocyte apoptosis. J Biol Chem 281: 23150–23166, 2006.[Abstract/Free Full Text]
243. Reinehr R, Becker S, Hongen A, Haussinger D. The Src family kinase Yes triggers hyperosmotic activation of the epidermal growth factor receptor and CD95. J Biol Chem 279: 23977–23987, 2004.[Abstract/Free Full Text]
244. Reinehr R, Graf D, Fischer R, Schliess F, Haussinger D. Hyperosmolarity triggers CD95 membrane trafficking and sensitizes rat hepatocytes toward CD95L-induced apoptosis. Hepatology 36: 602–614, 2002.[CrossRef][Web of Science][Medline]
245. Rim JS, Tanawattanacharoen S, Takenaka M, Handler JS, Kwon HM. The canine sodium/myo-inositol cotransporter gene: structural organization and characterization of the promoter. Arch Biochem Biophys 341: 193–199, 1997.[CrossRef][Web of Science][Medline]
246. Rittner HL, Hafner V, Klimiuk PA, Szweda LI, Goronzy JJ, Weyand CM. Aldose reductase functions as a detoxification system for lipid peroxidation products in vasculitis. J Clin Invest 103: 1007–1013, 1999.[Web of Science][Medline]
247. Rizoli SB, Rotstein OD, Kapus A. Cell volume-dependent regulation of L-selectin shedding in neutrophils. A role for p38 mitogen-activated protein kinase. J Biol Chem 274: 22072–22080, 1999.[Abstract/Free Full Text]
248. Robbins E, Pederson T, Klein P. Comparison of mitotic phenomena and effects induced by hypertonic solutions in HeLa cells. J Cell Biol 44: 400–416, 1970.[Abstract/Free Full Text]
249. Rodriguez I, Holloschi A, Kaszkin M, Cheng H, Kabsch K, Hafner M, Alonso A. Activation of phospholipase C-gamma1 in human keratinocytes by hyperosmolar shock without enzyme phosphorylation. Arch Dermatol Res 295: 490–497, 2004.[CrossRef][Web of Science][Medline]
250. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273: 5858–5868, 1998.[Abstract/Free Full Text]
251. Roger F, Martin PY, Rousselot M, Favre H, Feraille E. Cell shrinkage triggers the activation of mitogen-activated protein kinases by hypertonicity in the rat kidney medullary thick ascending limb of the Henle's loop. Requirement of p38 kinase for the regulatory volume increase response. J Biol Chem 274: 34103–34110, 1999.[Abstract/Free Full Text]
252. Roig J, Huang Z, Lytle C, Traugh JA. p21-activated protein kinase gamma-PAK is translocated and activated in response to hyperosmolarity. Implication of Cdc42 and phosphoinositide 3-kinase in a two-step mechanism for gamma-PAK activation. J Biol Chem 275: 16933–16940, 2000.[Abstract/Free Full Text]
253. Rosette C, Karin M. Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors. Science 274: 1194–1197, 1996.[Abstract/Free Full Text]
254. Saborio JL, Pong SS, Koch G. Selective and reversible inhibition of initiation of protein synthesis in mammalian cells. J Mol Biol 85: 195–211, 1974.[CrossRef][Web of Science][Medline]
255. Sands JM. Molecular approaches to urea transporters. J Am Soc Nephrol 13: 2795–2806, 2002.[Abstract/Free Full Text]
256. Sanguinetti AR, Cao H, Corley MC. Fyn is required for oxidative- and hyperosmotic-stress-induced tyrosine phosphorylation of caveolin-1. Biochem J 376: 159–168, 2003.[CrossRef][Web of Science][Medline]
257. Santos BC, Chevaile A, Hebert MJ, Zagajeski J, Gullans SR. A combination of NaCl and urea enhances survival of IMCD cells to hyperosmolality. Am J Physiol Renal Physiol 274: F1167–F1173, 1998.[Abstract/Free Full Text]
258. Santos BC, Chevaile A, Kojima R, Gullans SR. Characterization of the Hsp110/SSE gene family response to hyperosmolality and other stresses. Am J Physiol Renal Physiol 274: F1054–F1061, 1998.[Abstract/Free Full Text]
259. Santos BC, Pullman JM, Chevaile A, Welch WJ, Gullans SR. Chronic hyperosmolarity mediates constitutive expression of molecular chaperones and resistance to injury. Am J Physiol Renal Physiol 284: F564–F574, 2003.[Abstract/Free Full Text]
260. Santos BC, Pullman JM, Chevaile A, Welch WJ, Gullans SR. Chronic hyperosmolarity mediates constitutive expression of molecular chaperones and resistance to injury. Am J Physiol Renal Physiol 284: F564–F574, 2003.[Abstract/Free Full Text]
261. Schaffer S, Takahashi K, Azuma J. Role of osmoregulation in the actions of taurine. Amino Acids 19: 527–546, 2000.[CrossRef][Web of Science][Medline]
262. Schliess F, Heinrich S, Haussinger D. Hyperosmotic induction of the mitogen-activated protein kinase phosphatase MKP-1 in H4IIE rat hepatoma cells. Arch Biochem Biophys 351: 35–40, 1998.[CrossRef][Web of Science][Medline]
263. Shapiro L, Dinarello CA. Hyperosmotic stress as a stimulant for proinflammatory cytokine production. Exp Cell Res 231: 354–362, 1997.[CrossRef][Web of Science][Medline]
264. Sheen MR, Kim SW, Jung JY, Ahn JY, Rhee JG, Kwon HM, Woo SK. Mre11-Rad50-Nbs1 complex is activated by hypertonicity. Am J Physiol Renal Physiol 291: F1014–F1020, 2006.[Abstract/Free Full Text]
265. Sheikh-Hamad D, Di Mari J, Suki WN, Safirstein R, Watts BA III, Rouse D. p38 kinase activity is essential for osmotic induction of mRNAs for HSP70 and transporter for organic solute betaine in Madin-Darby canine kidney cells. J Biol Chem 273: 1832–1837, 1998.[Abstract/Free Full Text]
266. Sheikh-Hamad D, Ferraris JD, Dragolovich J, Preuss HG, Burg MB, Garcia-Perez A. CD9 antigen mRNA is induced by hypertonicity in two renal epithelial cell lines. Am J Physiol Cell Physiol 270: C253–C258, 1996.[Abstract/Free Full Text]
267. Sheikh-Hamad D, Nadkarni V, Choi YJ, Truong LD, Wideman C, Hodjati R, Gabbay KH. Cyclosporine A inhibits the adaptive responses to hypertonicity: a potential mechanism of nephrotoxicity. J Am Soc Nephrol 12: 2732–2741, 2001.[Abstract/Free Full Text]
268. Sheikh-Hamad D, Rouse D, Yang Y. Regulation of stanniocalcin in MDCK cells by hypertonicity and extracellular calcium. Am J Physiol Renal Physiol 278: F417–F424, 2000.[Abstract/Free Full Text]
269. Sheikh-Hamad D, Suki WN, Zhao W. Hypertonic induction of the cell adhesion molecule beta 1-integrin in MDCK cells. Am J Physiol Cell Physiol 273: C902–C908, 1997.[Abstract/Free Full Text]
270. Sheikh-Hamad D, Youker K, Truong LD, Nielsen S, Entman ML. Osmotically relevant membrane signaling complex: association between HB-EGF, beta(1)-integrin, CD9 in mTAL. Am J Physiol Cell Physiol 279: C136–C146, 2000.[Abstract/Free Full Text]
271. Shen MR, Chou CY, Hsu KF, Ellory JC. Osmotic shrinkage of human cervical cancer cells induces an extracellular Cl^–-dependent nonselective cation channel, which requires p38 MAPK. J Biol Chem 277: 45776–45784, 2002.[Abstract/Free Full Text]
272. Shim EH, Kim JI, Bang ES, Heo JS, Lee JS, Kim EY, Lee JE, Park WY, Kim SH, Kim HS, Smithies O, Jang JJ, Jin DI, Seo JS. Targeted disruption of hsp70.1 sensitizes to osmotic stress. EMBO Rep 3: 857–861, 2002.[CrossRef][Web of Science][Medline]
273. Smardo FL Jr, Burg MB, Garcia-Perez A. Kidney aldose reductase gene transcription is osmotically regulated. Am J Physiol Cell Physiol 262: C776–C782, 1992.[Abstract/Free Full Text]
274. Smith GC, Jackson SP. The DNA-dependent protein kinase. Genes Dev 13: 916–934, 1999.[Free Full Text]
275. Soleimani M, Singh G, Bizal GL, Gullans SR, McAteer JA. Na⁺/H⁺ exchanger isoforms NHE-2 and NHE-1 in inner medullary collecting duct cells. Expression, functional localization, differential regulation. J Biol Chem 269: 27973–27978, 1994.[Abstract/Free Full Text]
276. Soleimani M, Watts BA III, Singh G, Good DW. Effect of long-term hyperosmolality on the Na⁺/H⁺ exchanger isoform NHE-3 in LLC-PK1 cells. Kidney Int 53: 423–431, 1998.[CrossRef][Web of Science][Medline]
277. Spycher SE, Tabataba-Vakili S, O'Donnell VB, Palomba L, Azzi A. Aldose reductase induction: a novel response to oxidative stress of smooth muscle cells. FASEB J 11: 181–188, 1997.[Abstract]
278. Srivastava S, Chandra A, Bhatnagar A, Srivastava SK, Ansari NH. Lipid peroxidation product, 4-hydroxynonenal and its conjugate with GSH are excellent substrates of bovine lens aldose reductase. Biochem Biophys Res Commun 217: 741–746, 1995.[CrossRef][Web of Science][Medline]
279. Stadtman ER, Levine RL. Protein oxidation. Ann NY Acad Sci 899: 191–208, 2000.[Web of Science][Medline]
280. Steinbrecher KA, Rudolph JA, Luo G, Cohen MB. Coordinate upregulation of guanylin and uroguanylin expression by hypertonicity in HT29–18-N2 cells. Am J Physiol Cell Physiol 283: C1729–C1737, 2002.[Abstract/Free Full Text]
281. Storm R, Klussmann E, Geelhaar A, Rosenthal W, Maric K. Osmolality and solute composition are strong regulators of AQP2 expression in renal principal cells. Am J Physiol Renal Physiol 284: F189–F198, 2003.[Abstract/Free Full Text]
282. Street TO, Bolen DW, Rose GD. A molecular mechanism for osmolyte-induced protein stability. Proc Natl Acad Sci USA 103: 13997–14002, 2006.[Abstract/Free Full Text]
283. Strotmann R, Harteneck C, Nunnenmacher K, Schultz G, Plant TD. OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nat Cell Biol 2: 695–702, 2000.[CrossRef][Web of Science][Medline]
284. Stroud JC, Lopez-Rodriguez C, Rao A, Chen L. Structure of a TonEBP-DNA complex reveals DNA encircled by a transcription factor. Nat Struct Biol 9: 90–94, 2002.[CrossRef][Web of Science][Medline]
285. Sugiura T, Yamauchi A, Kitamura H, Matusoka Y, Horio M, Imai E, Hori M. Effects of hypertonic stress on transforming growth factor-beta activity in normal rat kidney cells. Kidney Int 53: 1654–1660, 1998.[CrossRef][Web of Science][Medline]
286. Takeda M, Homma T, Breyer MD, Horiba N, Hoover RL, Kawamoto S, Ichikawa I, Kon V. Volume and agonist-induced regulation of myosin light-chain phosphorylation in glomerular mesangial cells. Am J Physiol Renal Fluid Electrolyte Physiol 264: F421–F426, 1993.[Abstract/Free Full Text]
287. Takenaka M, Preston AS, Kwon HM, Handler JS. The tonicity-sensitive element that mediates increased transcription of the betaine transporter gene in response to hypertonic stress. J Biol Chem 269: 29379–29381, 1994.[Abstract/Free Full Text]
288. Tanaka K, Jay G, Isselbacher KJ. Expression of heat-shock and glucose-regulated genes: differential effects of glucose starvation and hypertonicity. Biochim Biophys Acta 950: 138–146, 1988.[Medline]
289. Terada Y, Tomita K, Homma MK, Nonoguchi H, Yang T, Yamada T, Yuasa Y, Krebs EG, Sasaki S, Marumo F. Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, S6 kinase by hyperosmolality in renal cells. J Biol Chem 269: 31296–31301, 1994.[Abstract/Free Full Text]
290. Tian W, Bonkovsky HL, Shibahara S, Cohen DM. Urea and hypertonicity increase expression of heme oxygenase-1 in murine renal medullary cells. Am J Physiol Renal Physiol 281: F983–F991, 2001.[Abstract/Free Full Text]
291. Tian W, Boss GR, Cohen DM. Ras signaling in the inner medullary cell response to urea and NaCl. Am J Physiol Cell Physiol 278: C372–C380, 2000.[Abstract/Free Full Text]
292. Tian W, Cohen DM. Signaling and gene regulation by urea in cells of the mammalian kidney medulla. Comp Biochem Physiol A Mol Integr Physiol 130: 429–436, 2001.[CrossRef][Medline]
293. Tian W, Cohen DM. Urea inhibits hypertonicity-inducible TonEBP expression and action. Am J Physiol Renal Physiol 280: F904–F912, 2001.[Abstract/Free Full Text]
294. Tian W, Cohen DM. Urea stress is more akin to EGF exposure than to hypertonic stress in renal medullary cells. Am J Physiol Renal Physiol 283: F388–F398, 2002.[Abstract/Free Full Text]
295. Tokiwa G, Dikic I, Lev S, Schlessinger J. Activation of Pyk2 by stress signals and coupling with JNK signaling pathway. Science 273: 792–794, 1996.[Abstract]
296. Tong EH, Guo JJ, Huang AL, Liu H, Hu CD, Chung SS, Ko BC. Regulation of nucleocytoplasmic trafficking of transcription factor OREBP/TonEBP/NFAT5. J Biol Chem 281: 23870–23879, 2006.[Abstract/Free Full Text]
297. Torbett NE, Casamassima A, Parker PJ. Hyperosmotic-induced protein kinase N 1 activation in a vesicular compartment is dependent upon Rac1 and 3-phosphoinositide-dependent kinase 1. J Biol Chem 278: 32344–32351, 2003.[Abstract/Free Full Text]
298. Uchida S, Garcia-Perez A, Murphy H, Burg M. Signal for induction of aldose reductase in renal medullary cells by high external NaCl. Am J Physiol Cell Physiol 256: C614–C620, 1989.[Abstract/Free Full Text]
299. Uchida S, Green N, Coon H, Triche T, Mims S, Burg M. High NaCl induces stable changes in phenotype and karyotype of renal cells in culture. Am J Physiol Cell Physiol 253: C230–C242, 1987.[Abstract/Free Full Text]
300. Uchida S, Kwon HM, Yamauchi A, Preston AS, Marumo F, Handler JS. Molecular cloning of the cDNA for an MDCK cell Na⁺- and Cl^–-dependent taurine transporter that is regulated by hypertonicity. Proc Natl Acad Sci USA 90: 7424, 1993.[Free Full Text]
301. Uchida S, Nakanishi T, Kwon HM, Preston AS, Handler JS. Taurine behaves as an osmolyte in Madin-Darby canine kidney cells. Protection by polarized, regulated transport of taurine. J Clin Invest 88: 656–662, 1991.[Web of Science][Medline]
302. Uchida S, Yamauchi A, Preston AS, Kwon HM, Handler JS. Medium tonicity regulates expression of the Na⁺- and Cl^–-dependent betaine transporter in Madin-Darby canine kidney cells by increasing transcription of the transporter gene. J Clin Invest 91: 1604–1607, 1993.[Web of Science][Medline]
303. Uhlik MT, Abell AN, Johnson NL, Sun W, Cuevas BD, Lobel-Rice KE, Horne EA, Dell'Acqua ML, Johnson GL. Rac-MEKK3-MKK3 scaffolding for p38 MAPK activation during hyperosmotic shock. Nat Cell Biol 5: 1104–1110, 2003.[CrossRef][Web of Science][Medline]
304. Umenishi F, Schrier RW. Identification and characterization of a novel hypertonicity-responsive element in the human aquaporin-1 gene. Biochem Biophys Res Commun 292: 771–775, 2002.[CrossRef][Web of Science][Medline]
305. Valkova N, Kultz D. Constitutive and inducible stress proteins dominate the proteome of the murine inner medullary collecting duct-3 (mIMCD3) cell line. Biochim Biophys Acta 1764: 1007–1020, 2006.[Medline]
306. Van der KJ, Beck M, Gray A, Downes CP. Distinct phosphatidylinositol 3-kinase lipid products accumulate upon oxidative and osmotic stress and lead to different cellular responses. J Biol Chem 274: 35963–35968, 1999.[Abstract/Free Full Text]
307. Wang X, Finegan KG, Robinson AC, Knowles L, Khosravi-Far R, Hinchliffe KA, Boot-Handford RP, Tournier C. Activation of extracellular signal-regulated protein kinase 5 downregulates FasL upon osmotic stress. Cell Death Differ. In press.
308. Wang Y, Ko BC, Yang JY, Lam TT, Jiang Z, Zhang J, Chung SK, Chung SS. Transgenic mice expressing dominant-negative osmoticresponse element-binding protein (OREBP) in lens exhibit fiber cell elongation defect associated with increased DNA breaks. J Biol Chem 280: 19986–19991, 2005.[Abstract/Free Full Text]
309. Ward IM, Chen J. Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J Biol Chem 276: 47759–47762, 2001.[Abstract/Free Full Text]
310. Warskulat U, Newsome W, Noe B, Stoll B, Haussinger D. Anisoosmotic regulation of hepatic gene expression. Biol Chem Hoppe-Seyler 377: 57–65, 1996.[Web of Science][Medline]
311. Wehner F, Olsen H, Tinel H, Kinne-Saffran E, Kinne RK. Cell volume regulation: osmolytes, osmolyte transport, signal transduction. Rev Physiol Biochem Pharmacol 148: 1–80, 2003.[Web of Science][Medline]
312. Wei LY, Roepe PD. Low external pH and osmotic shock increase the expression of human MDR protein. Biochemistry 33: 7229–7238, 1994.[CrossRef][Medline]
313. Wengler G, Wengler G. Medium hypertonicity and polyribosome structure in HeLa cells. The influence of hypertonicity of the growth medium on polyribosomes in HeLa cells. Eur J Biochem 27: 162–173, 1972.[Web of Science][Medline]
314. Werner E, Werb Z. Integrins engage mitochondrial function for signal transduction by a mechanism dependent on Rho GTPases. J Cell Biol 158: 357–368, 2002.[Abstract/Free Full Text]
315. Wiese S, Schliess F, Haussinger D. Osmotic regulation of MAP-kinase activities and gene expression in H4IIE rat hepatoma cells. Biol Chem 379: 667–671, 1998.[Web of Science][Medline]
316. Woo SK, Dahl SC, Handler JS, Kwon HM. Bidirectional regulation of tonicity-responsive enhancer binding protein in response to changes in tonicity. Am J Physiol Renal Physiol 278: F1006–F1012, 2000.[Abstract/Free Full Text]
317. Woo SK, Lee SD, Na KY, Park WK, Kwon HM. TonEBP/NFAT5 stimulates transcription of HSP70 in response to hypertonicity. Mol Cell Biol 22: 5753–5760, 2002.[Abstract/Free Full Text]
318. Woo SK, Maouyo D, Handler JS, Kwon HM. Nuclear redistribution of tonicity-responsive enhancer binding protein requires proteasome activity. Am J Physiol Cell Physiol 278: C323–C330, 2000.[Abstract/Free Full Text]
319. Wu KL, Khan S, Lakhe-Reddy S, Wang L, Jarad G, Miller RT, Konieczkowski M, Brown AM, Sedor JR, Schelling JR. Renal tubular epithelial cell apoptosis is associated with caspase cleavage of the NHE1 Na⁺/H⁺ exchanger. Am J Physiol Renal Physiol 284: F829–F839, 2003.[Abstract/Free Full Text]
320. Xiao Y, Weaver DT. Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells. Nucleic Acids Res 25: 2985–2991, 1997.[Abstract/Free Full Text]
321. Xu H, Tian W, Lindsley JN, Oyama TT, Capasso JM, Rivard CJ, Cohen HT, Bagnasco SM, Anderson S, Cohen DM. EphA2: expression in the renal medulla and regulation by hypertonicity and urea stress in vitro and in vivo. Am J Physiol Renal Physiol 288: F855–F866, 2005.[Abstract/Free Full Text]
322. Yamamoto M, Chen MZ, Wang YJ, Sun HQ, Wei Y, Martinez M, Yin HL. Hypertonic stress increases PI(4,5)P₂ levels by activating PIP5KIbeta. J Biol Chem 2006.
323. Yamauchi A, Uchida S, Kwon HM, Preston AS, Robey RB, Garcia-Perez A, Burg MB, Handler JS. Cloning of a Na⁺- and Cl^–-dependent betaine transporter that is regulated by hypertonicity. J Biol Chem 267: 649–652, 1992.[Abstract/Free Full Text]
324. Yamauchi A, Uchida S, Preston AS, Kwon HM, Handler JS. Hypertonicity stimulates transcription of gene for Na⁺-myo-inositol cotransporter in MDCK cells. Am J Physiol Renal Fluid Electrolyte Physiol 264: F20–F23, 1993.[Abstract/Free Full Text]
325. Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN. Living with water stress: evolution of osmolyte systems. Science 217: 1214–1222, 1982.[Abstract/Free Full Text]
326. Yang T, Schnermann JB, Briggs JP. Regulation of cyclooxygenase-2 expression in renal medulla by tonicity in vivo and in vitro. Am J Physiol Renal Physiol 277: F1–F9, 1999.[Abstract/Free Full Text]
327. Yang T, Terada Y, Nonoguchi H, Ujiie K, Tomita K, Marumo F. Effect of hyperosmolality on production and mRNA expression of ET-1 in inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 264: F684–F689, 1993.[Abstract/Free Full Text]
328. Yang T, Zhang A, Honeggar M, Kohan DE, Mizel D, Sanders K, Hoidal JR, Briggs JP, Schnermann JB. Hypertonic induction of COX-2 in collecting duct cells by reactive oxygen species of mitochondrial origin. J Biol Chem 280: 34966–34973, 2005.[Abstract/Free Full Text]
329. Yang XY, Zhang Z, Cohen DM. ERK activation by urea in the renal inner medullary mIMCD3 cell line. Am J Physiol Renal Physiol 277: F176–F185, 1999.[Abstract/Free Full Text]
330. Yu H, Li X, Marchetto GS, Dy R, Hunter D, Calvo B, Dawson TL, Wilm M, Anderegg RJ, Graves LM, Earp HS. Activation of a novel calcium-dependent protein-tyrosine kinase. Correlation with c-Jun N-terminal kinase but not mitogen-activated protein kinase activation. J Biol Chem 271: 29993–29998, 1996.[Abstract/Free Full Text]
331. Zablocki K. Hyperosmolality stimulates phospholipase A₂ activity in rabbit renal medulla and in Madin-Darby canine kidney (MDCK) cells. Int J Biochem Cell Biol 27: 1055–1063, 1995.[CrossRef][Web of Science][Medline]
332. Zablocki K, Miller SP, Garcia-Perez A, Burg MB. Accumulation of glycerophosphocholine (GPC) by renal cells: osmotic regulation of GPC:choline phosphodiesterase. Proc Natl Acad Sci USA 88: 7820–7824, 1991.[Abstract/Free Full Text]
333. Zaccheo O, Dinsdale D, Meacock PA, Glynn P. Neuropathy target esterase and its yeast homologue degrade phosphatidylcholine to glycerophosphocholine in living cells. J Biol Chem 279: 24024–24033, 2004.[Abstract/Free Full Text]
334. Zhang Z, Avraham H, Cohen DM. Urea and NaCl differentially regulate FAK and RAFTK/PYK2 in mIMCD3 renal medullary cells. Am J Physiol Renal Physiol 275: F447–F451, 1998.[Abstract/Free Full Text]
335. Zhang Z, Cai Q, Michea L, Dmitrieva N, Andrews P, Burg M. Proliferation and osmotic tolerance of renal inner medullary epithelial cells in vivo and in cell culture. Am J Physiol Renal Physiol 283: F203–F208, 2002.
336. Zhang Z, Cohen DM. NaCl but not urea activates p38 and jun kinase in mIMCD3 murine inner medullary cells. Am J Physiol Renal Fluid Electrolyte Physiol 271: F1234–F1238, 1996.[Abstract/Free Full Text]
337. Zhang Z, Dmitrieva NI, Park JH, Levine RL, Burg MB. High urea and NaCl carbonylate proteins in renal cells in culture and in vivo, and high urea causes 8-oxoguanine lesions in their DNA. Proc Natl Acad Sci USA 101: 9491–9496, 2004.[Abstract/Free Full Text]
338. Zhang Z, Ferraris J, Irarrazabal CE, Dmitireva NI, Park JH, Burg MB. Ataxia-telangiectasia mutated (ATM), a DNA damage-inducible kinase, contributes to high NaCl-induced nuclear localization of the transcription factor TonEBP/OREBP. Am J Physiol Renal Physiol 289: F506–F511, 2005.[Abstract/Free Full Text]
339. Zhang Z, Tian W, Cohen DM. Urea protects from the proapoptotic effect of NaCl in renal medullary cells. Am J Physiol Renal Physiol 279: F345–F352, 2000.[Abstract/Free Full Text]
340. Zhang Z, Yang XY, Cohen DM. Urea-associated oxidative stress and Gadd153/CHOP induction. Am J Physiol Renal Physiol 276: F786–F793, 1999.[Abstract/Free Full Text]
341. Zhang Z, Yang XY, Soltoff SP, Cohen DM. PI3K signaling in the murine kidney inner medullary cell response to urea. Am J Physiol Renal Physiol 278: F155–F164, 2000.[Abstract/Free Full Text]
342. Zhao H, Tian W, Tai C, Cohen DM. Hypertonic induction of COX-2 expression in renal medullary epithelial cells requires transactivation of the EGFR. Am J Physiol Renal Physiol 285: F281–F288, 2003.[Abstract/Free Full Text]
343. Zhao H, Tian W, Xu H, Cohen DM. Urea signalling to immediate-early gene transcription in renal medullary cells requires transactivation of the epidermal growth factor receptor. Biochem J 370: 479–487, 2003.[CrossRef][Web of Science][Medline]
344. Zhou B, Ann DK, Li X, Kim KJ, Lin H, Minoo P, Crandall ED, Borok Z. Hypertonic induction of aquaporin-5: novel role of hypoxia inducible factor-1. Am J Physiol Cell Physiol 292: C1280–C1290, 2007.[Abstract/Free Full Text]
345. Zhou BB, Elledge SJ. The DNA damage response: putting checkpoints in perspective. Nature 408: 433–439, 2000.[CrossRef][Medline]
346. Zhou C, Cammarata PR. Cloning the bovine Na⁺/myo-inositol cotransporter gene and characterization of an osmotic responsive promoter. Exp Eye Res 65: 349–363, 1997.[CrossRef][Web of Science][Medline]
347. Zhou X, Ferraris JD, Burg MB. Mitochondrial reactive oxygen species contribute to high NaCl-induced activation of the transcription factor TonEBP/OREBP. Am J Physiol Renal Physiol 290: F1169–F1176, 2006.[Abstract/Free Full Text]
348. Zhou X, Ferraris JD, Cai Q, Agarwal A, Burg MB. Increased reactive oxygen species contribute to high NaCl-induced activation of the osmoregulatory transcription factor TonEBP/OREBP. Am J Physiol Renal Physiol 289: F377–F385, 2005.[Abstract/Free Full Text]
349. Zhu J, Petersen S, Tessarollo L, Nussenzweig A. Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice. Curr Biol 11: 105–109, 2001.[CrossRef][Web of Science][Medline]
350. Zhuang S, Hirai SI, Ohno S. Hyperosmolality induces activation of cPKC and nPKC, a requirement for ERK1/2 activation in NIH/3T3 cells. Am J Physiol Cell Physiol 278: C102–C109, 2000.[Abstract/Free Full Text]
351. Zou AP, Billington H, Su N, Cowley AW Jr. Expression and actions of heme oxygenase in the renal medulla of rats. Hypertension 35: 342–347, 2000.[Abstract/Free Full Text]
352. Zou AP, Li N, Cowley AW Jr. Production and actions of superoxide in the renal medulla. Hypertension 37: 547–553, 2001.[Abstract/Free Full Text]

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