The interstitium of the renal medulla is hypertonic, imposing deleterious effects on local cells. At the same time, the hypertonicity provides osmotic gradient for water reabsorption and is a local signal for tissue-specific gene expression and differentiation of the renal medulla, which is a critical organ for water homeostasis.
Ambient tonicity is a powerful signal for gene expression and differentiation in addition to its osmotic action.
Water is the most abundant molecule in the body—60% by weight. Since the majority of biochemical reactions take place in water, maintenance of optimal volume and composition—water homeostasis—becomes critical. It is no wonder mammals, like other terrestrial animals, possess powerful and elaborate mechanisms for water homeostasis. Mammalian kidneys have the ability to adjust the urine output in response to a wide range of water intake by producing from very dilute urine, below 100 mosmol/kg, to highly concentrated urine, over 4,000 mosmol/kg in case of laboratory mice and rats, without changing the amount of total solute excretion. Remarkably, the interstitium of the renal medulla stays hypertonic throughout the dramatic fluctuations in the urinary osmolality. It is safe to state that the renal medullary cells are constantly bathed in variable but hypertonic interstitial solution. Traditionally, the term “hypertonicity” has been often used in conjunction with “stress,” since much of the interest has been focused on the detrimental action of hypertonicity. In this review, salubrious action of hypertonicity in the renal medulla will be discussed. Hypertonicity is an important local signal for the differentiation and maintenance of the renal medulla in addition to its role in the osmotic action in the urinary concentration. In genetically modified mice and in certain pathological conditions in which the medullary hypertonicity is compromised, the renal medulla loses tissue-specific gene expression profile, highlighting the importance of local hypertonicity in the maintenance of the renal medulla.
Definition of Hypertonicity
Operation of the urinary concentrating mechanism produces hyperosmolality in the interstitial fluid of the renal medulla. Osmolality of the renal medulla routinely reaches over 1,000 mosmol/kg in humans and 3,000 mosmol/kg in rodents. The hyperosmolality is due to accumulation of salt and urea. Salt and urea differ in their osmotic action. At the cellular level, salt is an effective solute, whereas urea is not. This means that, when the ambient osmolality is raised by addition of salt, cells shrink due to osmosis (movement of water across the plasma membrane). Urea has high membrane permeability comparable to water and thus does not induce significant osmosis. In the renal medulla, where complex arrangements of tubules, some of which have regulated urea permeability, in combination with dynamics of counter current flow of tubular fluids confer osmotic effectiveness to urea for water reabsorption. In this discussion, however, we will ignore this and define hypertonicity as hyperosmotic salt because we will focus on cellular effects.
Why is Hypertonicity Bad?
When cells are exposed to a hypertonic solution, cells loose water rapidly due to osmosis. Two immediate consequences of exposure to hypertonicity are 1) reduced cell volume and 2) increased ionic strength. It has been proposed that these changes perturb protein function due to macromolecular crowding (43) induced by cell shrinkage and elevated ionic strength (61). This concept was recently proved in an elegant body of work using the model organism C. elegans (5). In this study, a combination of the genome-wide RNAi screening and sophisticated cell biological and biophysical assays was used to show that hypertonicity induced protein damage (misfolding and aggregation) and that accumulation of damaged proteins caused death. Although the C. elegans study provide a proof-of-concept that hypertonicity damages proteins, whether the same mechanism causes cell death in the mammalian renal medullary cells is not known. What is clear is that hypertonicity also kills mammalian cells in a dose-dependent manner (42). Both apoptosis and necrosis are involved in the cell death.
TonEBP is a Master Regulator in Protection from Hyperosmolality in the Renal Medulla: Hypertonicity and Urea
All organisms except the extreme halophilic archebacteria use organic osmolytes for survival when challenged with hypertonic conditions (61). Organic osmolytes are polyhydric alcohols, amino acids and their derivatives, and methylamines. Unlike electrolytes, organic osmolytes are compatible with protein, i.e., osmolytes do not disrupt protein structure and function. Cellular accumulation of organic osmolytes removes the two sources of stress by hyper-tonicity—cell shrinkage and elevated ionic strength; rising concentration of organic osmolytes restores volume by osmosis and lowers the cellular ionic strength. C. elegans tolerate extreme hypertonicity when they are first allowed to accumulate glycerol, the major organic osmolyte for this animal, in moderate hyper-tonicity (28). Likewise, slow increase in ambient tonicity allows mammalian cells to adapt to extreme hypertonicity because cellular accumulation of organic osmolytes occurs over the course of many hours. The process is slow because stimulation of gene expression is involved. In mammalian cells, the transcription factor named TonEBP (tonicity-responsive enhancer binding protein) is the master transcriptional regulator for the cellular accumulation of organic osmolytes (45). While TonEBP is active under isotonic conditions, its activity increases when ambient tonicity rises (see below for more). TonEBP promotes cellular accumulation of the major organic osmolytes in the renal medulla by transcriptional stimulation of plasma membrane transporters—sodium/myoinositol cotransperter (SMIT), sodium/chloride/betaine cotransporter (BGT1), and sodium/chloride/taurine cotransporter (TauT)—and rate-limiting biosynthetic enzymes—aldose reductase (AR) for sorbitol and neuropathy target esterase (NTE, a phospholipase B) for glycerophosphorylcholine (FIGURE 1⇓) (11, 17, 44, 47, 58). The organic osmolytes—inositol, betaine, taurine, sorbitol, and glycerophosphorylcholine—are trapped inside the cell once they are transported or synthesized because they have low membrane permeability.
In C. elegans, induction of the gpdh genes that encodes glycerol 3-phosphate dehydrogenase is the key event in the hypertonicity-induced glycerol accumulation (29). Responsible transcription factor for the induction of gpdh genes is not known. TonEBP is not involved in the regulation because tonicity-responsive form of TonEBP is found only in vertebrates (26) and there is no TonEBP homolog in the nematode (14). Despite the lack of TonEBP in the nematode, it is possible that the mammals and the nematode share a signaling pathway conveying the hypertonicity signal to the stimulation of transcription.
Urea also causes cell death when its concentration exceeds 300 mM (42). Interestingly, TonEBP also plays a role in protection from the deleterious effects of urea by stimulating the expression of heat shock protein 70 (HSP70) (49). TonEBP binds to the promoter of the HSP70 gene in sites distinct from heat shock factor 1 and stimulates transcription of HSP70 (60). Every single cell in the renal medulla expresses high levels of TonEBP (4, 30). Genetic inhibition of TonEBP, either by gene deletion (36) or by overexpression of dominant negative form of TonEBP (27), results in severe renal medullary atrophy because of failure to adapt to hypertonicity and urea. These animals suffer from life-threatening dehydration and fail to thrive as they loose water via urine due to the lack of urinary concentrating ability. TonEBP is a master regulator in cellular protection from the harmful effects of hyperosmolality in the renal medulla (FIGURE 1⇑).
TonEBP Contributes to the Urinary Concentration Independent of Vasopressin
Although the antidiuretic hormone vasopressin is the major regulator of the urinary concentration by virtue of stimulating expression of aquaporin-2 (AQP2) water channels (50) and the UT-A urea transporters (22), there clearly is a vasopressin-independent component. For example, in Brattleboro rats, which lack endogenous vasopressin, expression of AQP2 is stimulated by manipulations that raise the medullary tonicity (32). In primary cultures (55) and established lines (15) of collecting duct principal cells, AQP2 expression increases in response to an increase in medium tonicity in a manner independent of vasopressin. There is a binding site for TonEBP in the promoter region of the AQP2 that mediates transcriptional stimulation in response to hypertonicity (16). Likewise, the promoter of the UT-A urea transporter gene is also stimulated by TonEBP (48). It should be pointed out that, throughout the collecting duct where AQP2 and UT-A are expressed, TonEBP is highly expressed, including the cortical segments (4, 30). In TonEBP-deficient transgenic mouse models, expression of AQP2 and UT-A is severely reduced (27, 36). Thus TonEBP is involved in the urinary concentrating process independent of vasopressin (FIGURE 1⇑).
Hypertonicity is a Local Signal for Differentiation and Maintenance of the Renal Medulla
As discussed above, the two major solutes in the renal medulla—salt and urea—differ in their osmotic action. Their effects on the functional and morphological differentiation of the renal medulla are also distinct. Urea concentration in the renal medulla can be easily manipulated without obvious or detrimental effects on the renal medulla. For example, low protein diet (via reducing the production of urea) (23) or increased water intake (via reducing circulating levels of vasopressin) (53) is quite effective in dramatically reducing the urea concentration in the renal medulla. Genetic deletion of the UT-A1 and UT-A3 urea transporters also has the same effect (9) since these proteins are the major route of urea reabsorption in the inner medulla. Lowering the urea concentration in the renal medulla does not affect the morphology or function of the renal medulla other than reducing urinary concentrating ability.
In contrast, the hypertonic salt concentration in the medullary interstitium is difficult to change. It is impossible to lower the interstitial hypertonicity on a long-term basis without affecting the integrity of the renal medulla. For example, a dramatic water diuresis with a 10-fold increase in the urine flow for several days does not affect the interstitial sodium concentration in the renal medulla (53, 54). Loop diuretics work acutely, but its effectiveness decreases over time because the kidney is vigorously adapting to the salt loss (7). For chronic reduction of the interstitial sodium concentration, life-threatening maneuvers such as continuous administration of high doses of loop diuretics are required (54). In mouse models of Bartter’s syndrome made by deletion of the gene encoding the Na-K-2Cl cotransporter type 2 (NKCC2) (56) or the ROMK potassium channel (37), renal medulla cannot be examined due to rampant hydronephrosis. There are, however, mouse models made by deletion of the AQP1 (39) or ClC-K1 gene (1) that exhibit significant decreases in the interstitial sodium concentration while maintaining intact renal medulla. In these animals, gene expression profile in the renal medulla is clearly altered (46), providing strong evidence that the local hypertonicity is an important signal for the medullary-specific gene expression. For example, expression of UT-A1 is dramatically decreased in these animals, most likely due to reduced activity of TonEBP. Another example is that those proteins normally enriched in the renal cortex such as the β- and γ-subunits of the ENaC channels are enriched in the inner medulla of the AQP1- or ClC-K1-deficient mice. Likewise, in cyclosporin-induced nephropathy (33) and hypokalemic animals (20), reduced TonEBP expression and activity in the renal medulla are observed in correlation with downregulation of NKCC2.
The local hypertonicity in the renal medulla appears to form early in the renal development. In mouse, expression of NKCC2 starts around E14 and is followed by TonEBP expression (30). Expression of TonEBP target genes such as UT-A and AR starts after the initiation of TonEBP expression. When the neonatal pups are treated with loop diuretic to inhibit the activity of NKCC2, developmental processes of the inner medulla are blocked (30). These observations demonstrate that the differentiation of the renal medulla requires local hypertonicity. Thus the interstitial hypertonicity of the renal medulla is an important local signal not only for specific gene expression profile but also for development of the renal medulla.
Bidirectional Regulation of TonEBP by Ambient Tonicity
TonEBP is also known as NFAT5—nuclear factor of activated T cells. In T cells, TonEBP is markedly induced on T-cell receptor activation (57). TonEBP regulates cytokines such as TNF-α and lymphotoxin-β in T-cells (35). Not surprisingly, the cytokine production is stimulated by hypertonicity. Transgenic mice expressing inhibitory TonEBP in T cells (58) and TonEBP knockout mice (13) reveal that TonEBP is required for T-cell proliferation and adaptive immunity. Interestingly, lymphoid tissues such as thymus and spleen are moderately hypertonic by ~40 mosmol/kg (13). In addition, TonEBP is required for proliferation of T cells under the mild hypertonicity (13). This is achieved by inducing sodium-coupled amino acid transporter-2 (SNAT2), which mediates cellular accumulation of amino acids as organic osmolytes (58). Thus TonEBP is critical for cellular adaptation to both mild (lymphoid tissues) and extreme (renal medulla) hypertonicity by promoting cellular accumulation of distinct organic osmolytes.
TonEBP is abundant in organs that are isotonic (41, 57). In brain, TonEBP expression is limited to neurons (38). Interestingly, nuclear localization of neuronal TonEBP increases rapidly in response to hypernatremia (systemic hypertonicity) in association with induction of ~190 genes in the brain (38, 40). This suggests that TonEBP is involved in the neuronal adaptation to hypertonicity. In other organs, TonEBP is clearly active in isotonic conditions (Table 1⇓). For example, TonEBP is involved in the differentiation of skeletal muscle via its target genes such as Cyr61 (51). Cyr61 is also implicated in tumor metastasis of the gastric cancer (34) and possibly breast cancer (19). In monocytes, TonEBP stimulates the replication of the human immunodeficiency viruses (HIV-1) by binding to their long-terminal repeat (LTR) (52).
In cultured cells, TonEBP is active in isotonic conditions based on expression of its target genes. Furthermore, TonEBP responds bidirectionally to both an increase and a decrease in the ambient tonicity: TonEBP activity decreases when ambient tonicity is lowered, whereas it increases when ambient tonicity is elevated (59). Ambient tonicity is a rheostat that controls the activity of TonEBP (FIGURE 2⇓). The expression of TonEBP in isotonic organs and tissues indicates that tonicity, i.e., isotonicity, is a widely used signal for transcription.
Molecular Mechanism of TonEBP Regulation by Ambient Tonicity
At least three discrete pathways are involved in the bidirectional regulation of TonEBP: nuclear localization, transactivation (stimulation of RNA polymerase complex for inititation of transcription), and abundance. Although phosphorylation of TonEBP increases in response to hypertonicity (31), changes in phosphorylation in response to hypotonicity are not defined, and sites of phosphorylation are not known.
Nuclear localization of TonEBP is determined by ambient tonicity in a highly predictable manner. Over the range of tonicity from 135 to 500 mosmol/kg, 25–55% of cellular TonEBP is in the nucleus as a tight function of tonicity (26). Thus the range of regulation is twofold (25–55%) for the nuclear localization. This regulation is completely dependent on the single nuclear localization signal (NLS) near the NH2-terminus (FIGURE 3⇓). Inactivation of the NLS by site-directed mutagenesis obliterates the tonicity-dependent changes in the nuclear localization: 25% of the mutant TonEBP molecules with inactive NLS is in the nucleus regardless of the ambient tonicity. When short fragments of TonEBP containing the NLS are fused to constitutively cytoplasmic proteins such as a triplet of green fluorescent protein, the fusion proteins displays tonicity-dependent changes in nuclear localization (26). These observations demonstrate that the tonicity-dependent changes in the nuclear localization of TonEBP are autonomously controlled by the NLS without involving other domains of TonEBP. Phosphorylation does not play a role in the regulation of the NLS because site-directed mutations that eliminate potential phosphorylation sites in the NLS display normal tonicity regulation.
The transactivating activity of TonEBP is also bidirectionally regulated by changes in ambient tonicity (10). There are three activation domains—AD1, AD2, and AD3—that stimulate transcription (31) (FIGURE 3⇑). In addition, there are two modulation domains—MD1 and MD2—that do not stimulate transcription by themselves but enhance the activity of activation domains. All the activation and modulations domains act cooperatively to confer a tremendous transactivational potential to TonEBP. AD2 and MD1 are stimulated by hypertonicity. This is not accompanied by changes in phosphorylation of AD2 or MD1 (31). On the other hand, RNA helicase A (RHA) binds the DNA binding domain and inhibits the transactivation of TonEBP (6). Since the catalytic activity of RHA is not required for the inhibitory action, it is possible that RHA facilitates recruitment of transcriptional repressors to TonEBP.
Like the nuclear localization, the level of TonEBP expression changes as a function of ambient tonicity in a bidirectional manner (59). These changes are accompanied by parallel changes in mRNA abundance. It is not known whether transcription is involved. A recent study suggests the role of mRNA stability in the tonicity-dependent changes in the abundance of TonEBP mRNA (3).
Hypertonicity is essential for the development and function of the renal medulla. TonEBP is the key transcription factor that controls gene expression in response to cues from ambient tonicity. As such, TonEBP is a master regulator in the renal medulla not only in cellular protection from the harmful hyperosmolality but also in the urinary concentration and development of the renal medulla. Outside the kidney, TonEBP is active in isotonic tissues, contributing to a variety of physiological and pathophysiological processes.
This work is supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-42479 and DK-61677. S. W. Lim is supported by a fellowship from the National Kidney Foundation.
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