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Kidney Cell Survival in High Tonicity
Comp. Biochem. Physiol. Vol. 117A, No. 3, pp. 301–306, 1997 Copyright 1997 Elsevier Science Inc.
ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00267-8
Kidney Cell Survival in High Tonicity Joseph S. Handler and H. Moo Kwon Department of Medicine, Johns Hopkins University, Baltimore, MD 21205, U.S.A. ABSTRACT. The kidney medulla of mammals undergoes large changes in tonicity in parallel with the tonicity of the final urine that emerges from the kidney at the tip of the medulla. When the medulla is hypertonic, its cells accumulate the compatible osmolytes myo-inositol, betaine, taurine, sorbitol and glycerophosphorylcholine. The mechanisms by which the compatible osmolytes are accumulated have been explored extensively in kidneyderived cells in culture. Myo-inositol, betaine and taurine are accumulated by increased activity of specific sodium-coupled transporters, sorbitol by increased synthesis of aldose reductase that catalyses the synthesis of sorbitol from glucose. Glycerophosphorylcholine accumulates primarily because its degradation is reduced in cells in hypertonic medium. cDNAs for the cotransporters and for aldose reductase have been cloned and used to establish that hypertonicity increases the transcription of the genes for the cotransporters for myo-inositol, betaine and for aldose reductase. The region 5′ to the promoter of the gene for the betaine cotransporter and for aldose reductase confer osmotic responsiveness to a heterologous promoter. The 12-bp sequence responsible for the transcriptional response to hypertonicity has been identified in the 5′ region of the gene for the betaine cotransporter. comp biochem physiol 117A;3:301–306, 1997. 1997 Elsevier Science Inc. KEY WORDS. Betaine, MDCK cells, myo-inositol, glycerophosphorylcholine, inositol, sorbitol, taurine, PAPHT25 cells
INTRODUCTION When the mammalian kidney produces a concentrated urine, the driving force for extraction of water from the urine is the hypertonicity in the kidney medulla. Medullary hypertonicity is distributed along a gradient that is lowest in the slightly hypertonic outer medulla adjacent to the isotonic renal cortex and increases to a peak at the papillary tip from which the final urine emerges. Cells in the kidney medulla experience shifts in tonicity comparable with shifts experienced by lower organisms living in estuaries. The tonicity at the tip of the papilla ranges from isotonic, when the kidney is producing a dilute urine, to as high as 1 osmolal in humans and more than 5 osmolal in some desert species (34). The cells in the medulla use a strategy similar to that of the cells of salinity tolerant organisms for survival during shifts in tonicity. When the medulla is hypertonic, medullary cells accumulate small organic solutes that have been designated compatible solutes. High concentrations of compatible solutes, in contrast to high concentrations of potassium or sodium, do not perturb the activity of enzymes and other cell macromolecules (52). Compatible solutes found in high concentrations in the hypertonic renal medulla include sorbitol, myo-inositol, betaine, taurine, glycerAddress reprint requests to: Dr. J.S. Handler, 965 Ross Building, Johns Hopkins School of Medicine, 720 Ross Research Building, Baltimore, MD 21205. Tel. (410) 955-0575; Fax (410) 955-0485; e-mail [email protected]. Received 7 September 1995; accepted 19 December 1995.
ophosphorylcholine and to a lesser extent certain amino acids (9). Another important part of the urinary concentrating mechanism involves the accumulation of urea in the medulla, with a concentration gradient that also increases from outer medulla to a peak at the papillary tip. The perturbing effect of intracellular urea is counteracted by the accumulation of the methylamine glycerophosphorylcholine (5). Urea and counteracting osmolytes have been reviewed recently (6) and are not considered further here. In this review, we use the term tonicity rather than osmolarity to distinguish between osmotic activity due to solutes that are biologically effective in raising osmotic activity in the compartment on one side of a cell membrane and those solutes, such as urea and glycerol, that permeate animal cell membranes fairly rapidly, and therefore do not effectively raise osmotic activity in the compartment on one side of a cell membrane. After the initial demonstrations that the kidney medulla accumulates compatible osmolytes (2), most understanding of the process of their accumulation and its regulation has come from studies of two immortal lines of kidney-derived cells in culture. When grown in a hypertonic medium, PAPHT25 cells, derived from the outer surface of the rabbit renal medulla (41), accumulate myo-inositol, betaine, glycerophosphorylcholine and sorbitol (21); MDCK cells, derived from an unknown site in the kidney of a dog, accumulate myo-inositol, betaine, glycerophosphorylcholine (21) and taurine (44). When the accumulation of compatible osmolytes is prevented, the ability of those cells to form colonies
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in hypertonic medium is dramatically impaired (44,51). Other kidney-derived cell lines accumulate compatible osmolytes when shifted to a hypertonic medium (21,29) but have not been studied as intensively as PAP-HT25 and MDCK cells. In this review we focus on work with cultured cells and extensions of that work to the kidney in situ. ACCUMULATION OF COMPATIBLE OSMOLYTES BY TRANSPORT Myo-inositol, betaine and taurine are accumulated in hypertonic cells by an increase in their uptake by specific transporters coupled to and driven by the uptake of sodium down its electrochemical gradient. Myo-inositol and betaine accumulate to concentrations as high as 500 times their extracellular concentration (29,49), in large part because of the driving force that results from the ratio of substrate compatible osmolyte to coupled ions, 2 sodiums/1 myo-inositol and 2 sodiums, 1 chloride/1 betaine. In addition, plasma membranes are very impermeable to the compatible osmolytes, preventing their loss. The cotransporters for the three compatible osmolytes are located on the basolateral surface of MDCK cells (44,49). All compatible osmolytes are accumulated slowly in response to a shift from isotonic to hypertonic medium. The activity of each cotransporter reaches a peak in about 24 hr as does the intracellular concentration of each substrate (23,24,43,49). The likelihood that the prolonged time course to maximal activation of each cotransporter was the result of increased synthesis of the cotransporters in response to the increase in tonicity was supported by the demonstration that mRNA (poly A1-RNA) from hypertonic MDCK cells caused increased expression of each cotransporter after injection into Xenopus oocytes (16,30,43). Expression from an MDCK cell cDNA library subsequently led to the cloning of the cDNA for the myo-inositol cotransporter (SMIT, sodium/myo-inositol transporter) (17), the betaine cotransporter (BGT1, betaine gamma amino butyric acid transporter) (46) and the taurine cotransporter (NCT, Na/chloride-dependent taurine cotransporter) (42). Myo-Inositol Cotransporter Analysis of the SMIT cDNA indicated that it probably has 12 transmembrane domains, a site for glycosylation on an extracellular loop, several consensus sequences for phosphorylation by protein kinase A (PKA) and by protein kinase C (PKC) and is a member of the gene family that includes the sodium-coupled glucose transporter initially cloned from rabbit small intestines (10). Northern hybridization and ribonuclease protection assays confirmed the initial evidence that hypertonicity elicited an increase in the abundance of SMIT mRNA, and nuclear run-on assays established increased transcription as the cause of the increased SMIT mRNA abundance (Fig. 1) (50). When cells
FIG. 1. Time course of the change in rate of transcription of
the SMIT gene (d), SMIT mRNA abundance (s) and activity of the Na1 /myo-inositol cotransporter (h) after MDCK cells are shifted (A) from an isotonic medium to a hypertonic medium and (B) when MDCK cells adapted to a hypertonic medium are shifted to an isotonic medium.
that had been adapted to hypertonic medium (500 mosm) were shifted to isotonic medium (300 mosm), transcription rate and mRNA abundance fell with a half-time of about 4 hr, whereas the activity of the transporter fell off more slowly (50). Increased abundance of SMIT mRNA and increased uptake of myo-inositol have been demonstrated in several types of mammalian cells in culture when they are shifted to hypertonic medium (19,25,54) and in cells in the rat renal medulla when its tonicity becomes hypertonic (47,48). Betaine and Taurine Cotransporters The betaine cotransporter and the taurine cotransporter belong to the family of neurotransmitter transporter genes (36). In contrast to the other members of the gene family, the osmolyte cotransporters’ biological function is to raise the intracellular concentration of their substrate rather
Kidney Cell Survival in High Tonicity
than to lower their substrate’s extracellular concentration. Both cotransporters have 12 putative transmembrane domains, a consensus sequence for N-linked glycosylation on a putative extracellular loop and consensus sequences for phosphorylation by PKA and by PKC on putative intracellular loops. The increased activity of both cotransporters that is elicited in MDCK cells by hypertonicity is the result of an increase in the abundance of mRNA for each cotransporter (42,46). The increase in BGT1 mRNA abundance, like that of SMIT, is the result of an increase in the rate of transcription of the BGT1 gene, which reaches a peak about 16 hr after the switch to hypertonicity (45). The canine BGT1 gene has been cloned. It consists of 18 exons distributed over 28 kb. There are three independent promoters and alternative splicing, resulting in multiple isoforms of mRNA. All variations in mRNA sequence are confined to the 5′ untranslated region and do not affect the open reading frame (37). It is not clear what purpose is served by this complex arrangement at the 5′ end of the gene. Transcription from all three promoters is stimulated by hypertonicity. Analysis of the 5′ flanking region of the BGT1 gene revealed a regulatory sequence element named TonE (tonicity-responsive element). The element is located between 62 and 50 nucleotides 5′ to the first exon. Its sequence contains an inverted repeat. TonE has the characteristics of an enhancer that is activated by hypertonicity. In luciferase transfection assays in MDCK cell, the enhancer function of TonE is orientation independent and concatamerization dramatically potentiates its activity. Electrophoretic mobility shift assays using nuclear extracts from MDCK cells and DNA containing the TonE sequence demonstrate a shift in mobility after the DNA is exposed to nuclear proteins from hypertonic MDCK cells (38). The shift is presumably the result of specific binding of a protein, TonEBP (TonE binding protein), that is barely detectable in nuclear extracts from isotonic MDCK cells. TonEBP may well be a transcription factor. The clear increase in activity of TonEBP indicates that activation or increased synthesis of TonEBP is the key step in stimulation of transcription of BGT1 by hypertonicity. It will be interesting to identify the hypertonicity-sensitive enhancer element in the SMIT gene that we have recently cloned (our unpublished observations). There is no information regarding the mechanism by which the abundance of NCT mRNA is increased by hypertonicity. It is not an attractive subject to study because NTC mRNA and the activity of the taurine cotransporter increase only about 2-fold in MDCK cells in hypertonic medium (43). The myo-inositol, the betaine and the taurine cotransporters are all subject to post-translational regulation by protein kinases. Activation of PKA or of PKC causes inhibition of the myo-inositol and the betaine cotransporters. For both cotransporters, the inhibitory effects of activation of PKA and of PKC are not additive (27a). Activation of PKC
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inhibits the taurine cotransporter in kidney derived LLCPK1 cells (13). We are not aware of information regarding the effect of activation of PKA on the activity of the taurine cotransporter. ACCUMULATION OF COMPATIBLE OSMOLYTES BY SYNTHESIS Sorbitol Hypertonicity markedly increases the accumulation of sorbitol in PAP-HT25 cells (21). The accumulation of sorbitol is the consequence of the more rapid increase in transcription of the aldose reductase gene, accumulation of aldose reductase mRNA and synthesis of the enzyme (20,33). The rabbit aldose reductase gene has been cloned. There is a tonicity-sensitive element(s) in the 3.5 kb of DNA 5′ to the promoter (8). It will be of great interest to compare the tonicity-sensitive sequence of the aldose reductase gene with the TonE sequence of MDCK cells. Sorbitol is degraded to fructose by the enzyme sorbitol dehydrogenase. Tonicity does not affect the activity of sorbitol dehydrogenase in PAP-HT25 cells. In contrast, aldose reductase and sorbitol dehydrogenase are reciprocally regulated in the rat kidney medulla. Reduction in urinary and medullary tonicity reduces aldose reductase activity and increases sorbitol dehydrogenase activity, whereas increased urinary and medullary tonicity results in increased aldose reductase activity and reduced activity of sorbitol dehydrogenase (18). Glycerophosphorylcholine Glycerophosphorylcholine is a product of phosphatidylcholine in a reaction catalyzed by phospholipase activity that is non-arachidonyl selective (15). Although the synthesis of glycerophosphorylcholine from phosphatidylcholine increases in MDCK cells shifted to a medium made hypertonic by addition of NaCl, the increase in synthesis is slower than the increase in glycerophosphorylcholine concentration (15) and the increased synthetic rate is thought to be of minor importance except when MDCK cells are maintained for several days in hypertonic medium. The increase in glycerophosphorylcholine levels in hypertonic cells is primarily the result of reduced degradation of glycerophosphorylcholine by the enzyme glycerophosphorylcholine:choline phosphodiesterase. Reduced degradation has been demonstrated both by following the rate of degradation of radiolabeled glycerophosphorylcholine in MDCK cells (15) and by assaying enzyme activity from isotonic and hypertonic MDCK cells (15,53). Glycerophosphorylcholine:choline phosphodiesterase activity is inhibited as well in cells exposed to high concentrations of urea (15), which also accumulates in the renal medulla when it is producing a hypertonic urine. Thus, the major factor in accumulation of glycerophosphorylcholine in renal medullary cells is reduction of its rate of degradation. Although both betaine and
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glycerophosphorylcholine are methylamines that counteract the perturbing effect of high concentrations of urea (52), MDCK cells exposed to high concentrations of urea typically accumulate glycerophosphorylcholine rather than betaine, whereas they accumulate both methylamines when cultured in medium made hypertonic by addition of NaCl or raffinose (6). The interaction among accumulated compatible and counteracting osmolytes has been reviewed recently (6). Recognition of Hypertonicity and Signaling the Response There is little information available regarding how mammalian cells recognize hypertonicity and how the signal is transduced to the transcriptional machinery. When PAPHT25 cells were shifted to a hypertonic medium, the increase in aldose reductase activity correlated best with the increase in cell potassium concentration. That led to the concept that the signal for induction of the accumulation of the compatible osmolytes might be related to the concentration of cell potassium (40). The interactions among accumulated compatible and counteracting osmolytes are consonant with that proposal in that changes in the accumulation of one compatible osmolyte results in reciprocal change in the accumulation of another. Those relationships have been reviewed (6,9). Studies in yeast have revealed a signal transduction pathway linking the sensing of hypertonicity to the transcriptional response. Two proteins in this pathway have been identified: HOG1 and PBS2 are homologues of mitogenactivated protein kinase (MAP kinase) and MAP kinase kinase, respectively (4). When yeast cells are switched to hypertonic media, both kinases are immediately activated, resulting in the stimulation of transcription of glycerol-3phosphate dehydrogenase, the rate-limiting enzyme for synthesis of glycerol, the predominant osmolyte of yeast, in an as yet undetermined way (1). In mammalian cells, hypertonicity stimulates three cascades of MAP kinase homologues: ERKs (11,39), JNK1/SAP and p38 (28). In MDCK cells, the stimulation of ERKs by hypertonicity is PKC dependent (11,39). To test for the role of ERK kinase in the induction of the myo-inositol cotransporter by hypertonicity, we studied MDCK cells that had been incubated for a long time with a high concentration of an active phorbol ester, a condition in which PKC is downregulated. In cells lacking PKC, the induction of SMIT mRNA by hypertonicity was not impaired, indicating that the ERK pathway is not an important component of the induction of the cotransporter by hypertonicity (16a). At this point, it is not clear whether the JNK1/SAP or p38 pathway is involved in activation of the cotransporters for compatible osmolytes. Hypertonicity induces an increase in the abundance of mRNA for some previously recognized stress proteins, such as the heat shock protein HSP-70 and the immediate early gene transcription factors Egr-1 and c-fos within 2 hr of the
switch (7). The induction of HSP-70, like that of the betaine cotransporter, is attenuated by facilitating the ability of cells to accumulate betaine or myo-inositol (27,32). The role of the stress proteins in organic osmolyte accumulation remains to be established. EFFLUX OF COMPATIBLE OSMOLYTES In contrast to the slow onset of the accumulation of nonperturbing osmolytes when cells are shifted to a hypertonic medium, the loss of compatible osmolytes occurs rapidly when cells are shifted to a medium of lower tonicity (3,22). Efflux of compatible osmolytes has a time course and sensitivity to inhibitors resembling those of the electrolyte loss that occurs in almost all cells when they shifted from an isotonic to a hypotonic medium (35). The loss occurs within 15 min of the shift and results in cells returning to their original size (regulatory volume decrease) as water follows the efflux of cell solutes. The efflux pathway has not been identified definitively, but indirect evidence has led to the suggestion that it may be a channel, perhaps the volume sensitive chloride channel that is activated by cell swelling (14,26,31). There is direct evidence for conductance of taurine via that channel in swollen cells (12). The evidence for the swelling activated chloride channel serving as the egress pathway for organic osmolytes has been summarized recently (35). In conclusion, among the key issues remaining to be explored are the early steps in regulation of transcription of the genes for the transporters of compatible osmolytes and the gene for aldose reductase. As more tonicity-responsive transcriptional regulatory sequences are identified, it should become possible to identify the transacting factor(s) that bind to them. The evidence that hypertonicity activates MAP kinase related pathways in a variety of cells calls for further exploration of the role of that type of activation in regulation of the response to hypertonicity. References 1. Albertyn, J.; Hohmann, S.; Thevelein, J.M.; Prior, B.A. GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway. Mol. Cell. Biol. 14:4135–4144; 1994. 2. Bagnasco, S.M.; Balaban, R.; Fales, H.; Yang, Y.-M.; Burg, M.B. Predominant osmotically active organic solutes in rat and rabbit renal medullas. J. Biol. Chem. 261:5872–5877; 1986. 3. Bagnasco, S.M.; Murphy, H.R.; Bedford, J.J.; Burg, M.B. Osmoregulation by slow changes in aldose reductase and rapid changes in sorbitol efflux. Am. J. Physiol. 254:C768–C792; 1988. 4. Brewster, J.L.; de Valoir, T.; Dwyer, N.D.; Winter, E.; Gustin, M.C. An osmosensing signal transduction pathway in yeast. Science 259:1760–1763;1993. 5. Burg, M.B. Molecular basis for accumulation of compatible osmolytes in mammalian cells. In: Somero, E.A. (ed). Water and Life. Berlin, Heidelberg: Springer-Verlag; 1992:33–51.
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47. Yamauchi, A.; Nakanishi, T.; Takamitsu, Y.; Sugita, M.; Imai, E.; Noguchi, T.; Fujiwara, Y.; Kamada, T.; Ueda, N. In vivo osmoregulation of Na/myo-inositol cotransporter mRNA in rat kidney medulla. J. Am. Soc. Nephrol. 5:62–67;1994. 48. Yamauchi, A.; Miyai, A.; Shimada, S.; Minami, Y.; Tohyama, M.; Ueda, N. Localization and rapid regulation of Na1 /myoinositol cotransporter in rat kidney. J. Clin. Invest. 96:1195– 1201;1995. 49. Yamauchi, A.; Kwon, H.M.; Uchida, S.; Preston, A.S.; Handler, J.S. Myo-inositol and betaine transporters regulated by tonicity are basolateral in MDCK cells. Am. J. Physiol. 261: F197–F202;1991. 50. Yamauchi, A.; Uchida, S.; Preston, A.S.; Kwon, H.M.; Handler, J.S. Hypertonicity stimulates transcription of gene for Na1 –myo-inositol cotransporter in MDCK cells. Am. J. Physiol. 264:F20–F23;1993. 51. Yancey, P.H.; Burg, M.B.; Bagnasco, S.M. Effects of NaCl, glucose, and aldose reductase inhibitors on cloning efficiency of renal medullary cells. Am. J. Physiol. 258:C156–C163; 1990. 52. Yancey, P.H.; Clark, M.E.; Hand, S.C.; Bowlus, R.D.; Somero, G.N. Living with water stress: evolution of osmolyte system. Science 217:1214–1222;1982. 53. Zablocki, K.; Miller, S.P.F.; Garcia-Perez, A.; Burg, M.B. Accumulation of glycerophosphocholine (GPC) by renal cells: osmotic regulation of GPC: choline phosphodiesterase. Proc. Natl. Acad. Sci. USA 88:7820–7824;1991. 54. Zhou, C.; Chen, H.Q.; Reeves, R.; Agarwal, N.; Cammarata, P.R. Osmoregulatory alterations in myo-inositol uptake by bovine lens epithelial cells. Invest. Ophthal. Vis. Sci. 35:4118 – 4125;1994.
ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00267-8
Kidney Cell Survival in High Tonicity Joseph S. Handler and H. Moo Kwon Department of Medicine, Johns Hopkins University, Baltimore, MD 21205, U.S.A. ABSTRACT. The kidney medulla of mammals undergoes large changes in tonicity in parallel with the tonicity of the final urine that emerges from the kidney at the tip of the medulla. When the medulla is hypertonic, its cells accumulate the compatible osmolytes myo-inositol, betaine, taurine, sorbitol and glycerophosphorylcholine. The mechanisms by which the compatible osmolytes are accumulated have been explored extensively in kidneyderived cells in culture. Myo-inositol, betaine and taurine are accumulated by increased activity of specific sodium-coupled transporters, sorbitol by increased synthesis of aldose reductase that catalyses the synthesis of sorbitol from glucose. Glycerophosphorylcholine accumulates primarily because its degradation is reduced in cells in hypertonic medium. cDNAs for the cotransporters and for aldose reductase have been cloned and used to establish that hypertonicity increases the transcription of the genes for the cotransporters for myo-inositol, betaine and for aldose reductase. The region 5′ to the promoter of the gene for the betaine cotransporter and for aldose reductase confer osmotic responsiveness to a heterologous promoter. The 12-bp sequence responsible for the transcriptional response to hypertonicity has been identified in the 5′ region of the gene for the betaine cotransporter. comp biochem physiol 117A;3:301–306, 1997. 1997 Elsevier Science Inc. KEY WORDS. Betaine, MDCK cells, myo-inositol, glycerophosphorylcholine, inositol, sorbitol, taurine, PAPHT25 cells
INTRODUCTION When the mammalian kidney produces a concentrated urine, the driving force for extraction of water from the urine is the hypertonicity in the kidney medulla. Medullary hypertonicity is distributed along a gradient that is lowest in the slightly hypertonic outer medulla adjacent to the isotonic renal cortex and increases to a peak at the papillary tip from which the final urine emerges. Cells in the kidney medulla experience shifts in tonicity comparable with shifts experienced by lower organisms living in estuaries. The tonicity at the tip of the papilla ranges from isotonic, when the kidney is producing a dilute urine, to as high as 1 osmolal in humans and more than 5 osmolal in some desert species (34). The cells in the medulla use a strategy similar to that of the cells of salinity tolerant organisms for survival during shifts in tonicity. When the medulla is hypertonic, medullary cells accumulate small organic solutes that have been designated compatible solutes. High concentrations of compatible solutes, in contrast to high concentrations of potassium or sodium, do not perturb the activity of enzymes and other cell macromolecules (52). Compatible solutes found in high concentrations in the hypertonic renal medulla include sorbitol, myo-inositol, betaine, taurine, glycerAddress reprint requests to: Dr. J.S. Handler, 965 Ross Building, Johns Hopkins School of Medicine, 720 Ross Research Building, Baltimore, MD 21205. Tel. (410) 955-0575; Fax (410) 955-0485; e-mail [email protected]. Received 7 September 1995; accepted 19 December 1995.
ophosphorylcholine and to a lesser extent certain amino acids (9). Another important part of the urinary concentrating mechanism involves the accumulation of urea in the medulla, with a concentration gradient that also increases from outer medulla to a peak at the papillary tip. The perturbing effect of intracellular urea is counteracted by the accumulation of the methylamine glycerophosphorylcholine (5). Urea and counteracting osmolytes have been reviewed recently (6) and are not considered further here. In this review, we use the term tonicity rather than osmolarity to distinguish between osmotic activity due to solutes that are biologically effective in raising osmotic activity in the compartment on one side of a cell membrane and those solutes, such as urea and glycerol, that permeate animal cell membranes fairly rapidly, and therefore do not effectively raise osmotic activity in the compartment on one side of a cell membrane. After the initial demonstrations that the kidney medulla accumulates compatible osmolytes (2), most understanding of the process of their accumulation and its regulation has come from studies of two immortal lines of kidney-derived cells in culture. When grown in a hypertonic medium, PAPHT25 cells, derived from the outer surface of the rabbit renal medulla (41), accumulate myo-inositol, betaine, glycerophosphorylcholine and sorbitol (21); MDCK cells, derived from an unknown site in the kidney of a dog, accumulate myo-inositol, betaine, glycerophosphorylcholine (21) and taurine (44). When the accumulation of compatible osmolytes is prevented, the ability of those cells to form colonies
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in hypertonic medium is dramatically impaired (44,51). Other kidney-derived cell lines accumulate compatible osmolytes when shifted to a hypertonic medium (21,29) but have not been studied as intensively as PAP-HT25 and MDCK cells. In this review we focus on work with cultured cells and extensions of that work to the kidney in situ. ACCUMULATION OF COMPATIBLE OSMOLYTES BY TRANSPORT Myo-inositol, betaine and taurine are accumulated in hypertonic cells by an increase in their uptake by specific transporters coupled to and driven by the uptake of sodium down its electrochemical gradient. Myo-inositol and betaine accumulate to concentrations as high as 500 times their extracellular concentration (29,49), in large part because of the driving force that results from the ratio of substrate compatible osmolyte to coupled ions, 2 sodiums/1 myo-inositol and 2 sodiums, 1 chloride/1 betaine. In addition, plasma membranes are very impermeable to the compatible osmolytes, preventing their loss. The cotransporters for the three compatible osmolytes are located on the basolateral surface of MDCK cells (44,49). All compatible osmolytes are accumulated slowly in response to a shift from isotonic to hypertonic medium. The activity of each cotransporter reaches a peak in about 24 hr as does the intracellular concentration of each substrate (23,24,43,49). The likelihood that the prolonged time course to maximal activation of each cotransporter was the result of increased synthesis of the cotransporters in response to the increase in tonicity was supported by the demonstration that mRNA (poly A1-RNA) from hypertonic MDCK cells caused increased expression of each cotransporter after injection into Xenopus oocytes (16,30,43). Expression from an MDCK cell cDNA library subsequently led to the cloning of the cDNA for the myo-inositol cotransporter (SMIT, sodium/myo-inositol transporter) (17), the betaine cotransporter (BGT1, betaine gamma amino butyric acid transporter) (46) and the taurine cotransporter (NCT, Na/chloride-dependent taurine cotransporter) (42). Myo-Inositol Cotransporter Analysis of the SMIT cDNA indicated that it probably has 12 transmembrane domains, a site for glycosylation on an extracellular loop, several consensus sequences for phosphorylation by protein kinase A (PKA) and by protein kinase C (PKC) and is a member of the gene family that includes the sodium-coupled glucose transporter initially cloned from rabbit small intestines (10). Northern hybridization and ribonuclease protection assays confirmed the initial evidence that hypertonicity elicited an increase in the abundance of SMIT mRNA, and nuclear run-on assays established increased transcription as the cause of the increased SMIT mRNA abundance (Fig. 1) (50). When cells
FIG. 1. Time course of the change in rate of transcription of
the SMIT gene (d), SMIT mRNA abundance (s) and activity of the Na1 /myo-inositol cotransporter (h) after MDCK cells are shifted (A) from an isotonic medium to a hypertonic medium and (B) when MDCK cells adapted to a hypertonic medium are shifted to an isotonic medium.
that had been adapted to hypertonic medium (500 mosm) were shifted to isotonic medium (300 mosm), transcription rate and mRNA abundance fell with a half-time of about 4 hr, whereas the activity of the transporter fell off more slowly (50). Increased abundance of SMIT mRNA and increased uptake of myo-inositol have been demonstrated in several types of mammalian cells in culture when they are shifted to hypertonic medium (19,25,54) and in cells in the rat renal medulla when its tonicity becomes hypertonic (47,48). Betaine and Taurine Cotransporters The betaine cotransporter and the taurine cotransporter belong to the family of neurotransmitter transporter genes (36). In contrast to the other members of the gene family, the osmolyte cotransporters’ biological function is to raise the intracellular concentration of their substrate rather
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than to lower their substrate’s extracellular concentration. Both cotransporters have 12 putative transmembrane domains, a consensus sequence for N-linked glycosylation on a putative extracellular loop and consensus sequences for phosphorylation by PKA and by PKC on putative intracellular loops. The increased activity of both cotransporters that is elicited in MDCK cells by hypertonicity is the result of an increase in the abundance of mRNA for each cotransporter (42,46). The increase in BGT1 mRNA abundance, like that of SMIT, is the result of an increase in the rate of transcription of the BGT1 gene, which reaches a peak about 16 hr after the switch to hypertonicity (45). The canine BGT1 gene has been cloned. It consists of 18 exons distributed over 28 kb. There are three independent promoters and alternative splicing, resulting in multiple isoforms of mRNA. All variations in mRNA sequence are confined to the 5′ untranslated region and do not affect the open reading frame (37). It is not clear what purpose is served by this complex arrangement at the 5′ end of the gene. Transcription from all three promoters is stimulated by hypertonicity. Analysis of the 5′ flanking region of the BGT1 gene revealed a regulatory sequence element named TonE (tonicity-responsive element). The element is located between 62 and 50 nucleotides 5′ to the first exon. Its sequence contains an inverted repeat. TonE has the characteristics of an enhancer that is activated by hypertonicity. In luciferase transfection assays in MDCK cell, the enhancer function of TonE is orientation independent and concatamerization dramatically potentiates its activity. Electrophoretic mobility shift assays using nuclear extracts from MDCK cells and DNA containing the TonE sequence demonstrate a shift in mobility after the DNA is exposed to nuclear proteins from hypertonic MDCK cells (38). The shift is presumably the result of specific binding of a protein, TonEBP (TonE binding protein), that is barely detectable in nuclear extracts from isotonic MDCK cells. TonEBP may well be a transcription factor. The clear increase in activity of TonEBP indicates that activation or increased synthesis of TonEBP is the key step in stimulation of transcription of BGT1 by hypertonicity. It will be interesting to identify the hypertonicity-sensitive enhancer element in the SMIT gene that we have recently cloned (our unpublished observations). There is no information regarding the mechanism by which the abundance of NCT mRNA is increased by hypertonicity. It is not an attractive subject to study because NTC mRNA and the activity of the taurine cotransporter increase only about 2-fold in MDCK cells in hypertonic medium (43). The myo-inositol, the betaine and the taurine cotransporters are all subject to post-translational regulation by protein kinases. Activation of PKA or of PKC causes inhibition of the myo-inositol and the betaine cotransporters. For both cotransporters, the inhibitory effects of activation of PKA and of PKC are not additive (27a). Activation of PKC
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inhibits the taurine cotransporter in kidney derived LLCPK1 cells (13). We are not aware of information regarding the effect of activation of PKA on the activity of the taurine cotransporter. ACCUMULATION OF COMPATIBLE OSMOLYTES BY SYNTHESIS Sorbitol Hypertonicity markedly increases the accumulation of sorbitol in PAP-HT25 cells (21). The accumulation of sorbitol is the consequence of the more rapid increase in transcription of the aldose reductase gene, accumulation of aldose reductase mRNA and synthesis of the enzyme (20,33). The rabbit aldose reductase gene has been cloned. There is a tonicity-sensitive element(s) in the 3.5 kb of DNA 5′ to the promoter (8). It will be of great interest to compare the tonicity-sensitive sequence of the aldose reductase gene with the TonE sequence of MDCK cells. Sorbitol is degraded to fructose by the enzyme sorbitol dehydrogenase. Tonicity does not affect the activity of sorbitol dehydrogenase in PAP-HT25 cells. In contrast, aldose reductase and sorbitol dehydrogenase are reciprocally regulated in the rat kidney medulla. Reduction in urinary and medullary tonicity reduces aldose reductase activity and increases sorbitol dehydrogenase activity, whereas increased urinary and medullary tonicity results in increased aldose reductase activity and reduced activity of sorbitol dehydrogenase (18). Glycerophosphorylcholine Glycerophosphorylcholine is a product of phosphatidylcholine in a reaction catalyzed by phospholipase activity that is non-arachidonyl selective (15). Although the synthesis of glycerophosphorylcholine from phosphatidylcholine increases in MDCK cells shifted to a medium made hypertonic by addition of NaCl, the increase in synthesis is slower than the increase in glycerophosphorylcholine concentration (15) and the increased synthetic rate is thought to be of minor importance except when MDCK cells are maintained for several days in hypertonic medium. The increase in glycerophosphorylcholine levels in hypertonic cells is primarily the result of reduced degradation of glycerophosphorylcholine by the enzyme glycerophosphorylcholine:choline phosphodiesterase. Reduced degradation has been demonstrated both by following the rate of degradation of radiolabeled glycerophosphorylcholine in MDCK cells (15) and by assaying enzyme activity from isotonic and hypertonic MDCK cells (15,53). Glycerophosphorylcholine:choline phosphodiesterase activity is inhibited as well in cells exposed to high concentrations of urea (15), which also accumulates in the renal medulla when it is producing a hypertonic urine. Thus, the major factor in accumulation of glycerophosphorylcholine in renal medullary cells is reduction of its rate of degradation. Although both betaine and
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glycerophosphorylcholine are methylamines that counteract the perturbing effect of high concentrations of urea (52), MDCK cells exposed to high concentrations of urea typically accumulate glycerophosphorylcholine rather than betaine, whereas they accumulate both methylamines when cultured in medium made hypertonic by addition of NaCl or raffinose (6). The interaction among accumulated compatible and counteracting osmolytes has been reviewed recently (6). Recognition of Hypertonicity and Signaling the Response There is little information available regarding how mammalian cells recognize hypertonicity and how the signal is transduced to the transcriptional machinery. When PAPHT25 cells were shifted to a hypertonic medium, the increase in aldose reductase activity correlated best with the increase in cell potassium concentration. That led to the concept that the signal for induction of the accumulation of the compatible osmolytes might be related to the concentration of cell potassium (40). The interactions among accumulated compatible and counteracting osmolytes are consonant with that proposal in that changes in the accumulation of one compatible osmolyte results in reciprocal change in the accumulation of another. Those relationships have been reviewed (6,9). Studies in yeast have revealed a signal transduction pathway linking the sensing of hypertonicity to the transcriptional response. Two proteins in this pathway have been identified: HOG1 and PBS2 are homologues of mitogenactivated protein kinase (MAP kinase) and MAP kinase kinase, respectively (4). When yeast cells are switched to hypertonic media, both kinases are immediately activated, resulting in the stimulation of transcription of glycerol-3phosphate dehydrogenase, the rate-limiting enzyme for synthesis of glycerol, the predominant osmolyte of yeast, in an as yet undetermined way (1). In mammalian cells, hypertonicity stimulates three cascades of MAP kinase homologues: ERKs (11,39), JNK1/SAP and p38 (28). In MDCK cells, the stimulation of ERKs by hypertonicity is PKC dependent (11,39). To test for the role of ERK kinase in the induction of the myo-inositol cotransporter by hypertonicity, we studied MDCK cells that had been incubated for a long time with a high concentration of an active phorbol ester, a condition in which PKC is downregulated. In cells lacking PKC, the induction of SMIT mRNA by hypertonicity was not impaired, indicating that the ERK pathway is not an important component of the induction of the cotransporter by hypertonicity (16a). At this point, it is not clear whether the JNK1/SAP or p38 pathway is involved in activation of the cotransporters for compatible osmolytes. Hypertonicity induces an increase in the abundance of mRNA for some previously recognized stress proteins, such as the heat shock protein HSP-70 and the immediate early gene transcription factors Egr-1 and c-fos within 2 hr of the
switch (7). The induction of HSP-70, like that of the betaine cotransporter, is attenuated by facilitating the ability of cells to accumulate betaine or myo-inositol (27,32). The role of the stress proteins in organic osmolyte accumulation remains to be established. EFFLUX OF COMPATIBLE OSMOLYTES In contrast to the slow onset of the accumulation of nonperturbing osmolytes when cells are shifted to a hypertonic medium, the loss of compatible osmolytes occurs rapidly when cells are shifted to a medium of lower tonicity (3,22). Efflux of compatible osmolytes has a time course and sensitivity to inhibitors resembling those of the electrolyte loss that occurs in almost all cells when they shifted from an isotonic to a hypotonic medium (35). The loss occurs within 15 min of the shift and results in cells returning to their original size (regulatory volume decrease) as water follows the efflux of cell solutes. The efflux pathway has not been identified definitively, but indirect evidence has led to the suggestion that it may be a channel, perhaps the volume sensitive chloride channel that is activated by cell swelling (14,26,31). There is direct evidence for conductance of taurine via that channel in swollen cells (12). The evidence for the swelling activated chloride channel serving as the egress pathway for organic osmolytes has been summarized recently (35). In conclusion, among the key issues remaining to be explored are the early steps in regulation of transcription of the genes for the transporters of compatible osmolytes and the gene for aldose reductase. As more tonicity-responsive transcriptional regulatory sequences are identified, it should become possible to identify the transacting factor(s) that bind to them. The evidence that hypertonicity activates MAP kinase related pathways in a variety of cells calls for further exploration of the role of that type of activation in regulation of the response to hypertonicity. References 1. Albertyn, J.; Hohmann, S.; Thevelein, J.M.; Prior, B.A. GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway. Mol. Cell. Biol. 14:4135–4144; 1994. 2. Bagnasco, S.M.; Balaban, R.; Fales, H.; Yang, Y.-M.; Burg, M.B. Predominant osmotically active organic solutes in rat and rabbit renal medullas. J. Biol. Chem. 261:5872–5877; 1986. 3. Bagnasco, S.M.; Murphy, H.R.; Bedford, J.J.; Burg, M.B. Osmoregulation by slow changes in aldose reductase and rapid changes in sorbitol efflux. Am. J. Physiol. 254:C768–C792; 1988. 4. Brewster, J.L.; de Valoir, T.; Dwyer, N.D.; Winter, E.; Gustin, M.C. An osmosensing signal transduction pathway in yeast. Science 259:1760–1763;1993. 5. Burg, M.B. Molecular basis for accumulation of compatible osmolytes in mammalian cells. In: Somero, E.A. (ed). Water and Life. Berlin, Heidelberg: Springer-Verlag; 1992:33–51.
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24. Nakanishi, T.; Turner, R.J.; Brug, M.B. Osmoregulation of betaine transport in mammalian renal medullary cells. Am. J. Physiol. 258:F1061–F1067;1990. 25. Paredes, A.; McManus, M.; Kwon, H.M.; Strange, K. Osmoregulation of Na1-inositol cotransporter activity and mRNA levels in brain glial cells. Am. J. Physiol. 263:C1282–C1288; 1992. 26. Pasantes-Morales, H.; Murray, R.A.; Sanchez-Olea, R.; Moran, J. Regulatory volume decrease in cultured astrocytes II. Permeability pathway to amino acids and polyols. Am. Physiol. Soc. 266:C172–C178;1994. 27. Petronini, P.G.; DeAngelis, E.M.; Borghetti, A.F.; Wheeler, K.P. Effect of betaine on HSP70 expression and cell survival during adaptation to osmotic stress. Biochem. J. 293:553–558; 1993. 27a. Preston, A.S.; Yamauchi, A.; Kwon, H.M.; Handler, J.S. Activators of protein kinase A and of protein kinase C inhibit MDCK cell myo-inositol and betaine uptake. J. Am. Soc. Nephrol. 6:1559–1564;1995. 28. Raingeaud, J.; Gupta, S.; Rogers, J.S.; Dickens, M.; Han, J.; Ulevitch, R.J.; Davis, R.J. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J. Biol. Chem. 270:7420 –7426;1995. 29. Rauchman, M.I.; Nigam, S.K.; Delpire, E.; Gullans, S.R. An osmolytically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse. Am. J. Physiol. 265:F416– F424;1993. 30. Robey, R.B.; Kwon, H.M.; Handler, J.S.; Garcia-Perez, A.; Burg, M.B. Induction of glycinebetaine uptake into Xenopus oocytes by injection of poly(A)1 RNA from renal cells exposed to high extracellular NaCl. J. Biol. Chem. 266:10400 – 10405;1991. 31. Roy, G.; Malo, C. Activation of amino acid diffusion by a volume increase in cultured kidney (MDCK) cells. J. Membrane Biol. 130:83–90;1992. 32. Sheikh-Hamad, D.; Garcia-Perez, A.; Ferraris, J.D.; Peters, E.M.; Burg, M.B. Induction of gene expression by heat versus osmotic stress. Am. J. Physiol. 267:F28 –F34;1994. 33. Smardo, F.L., Jr.; Burg, M.B.; Garcia-Perez, A. Kidney aldose reductase gene transcription is osmotically regulated. Am. J. Physiol. 262:C776–C782;1992. 34. Stephenson, J.L. Renal concentrating mechanism. Federation Proc. 42:2377–2378;1983. 35. Strange, K.; Jackson, P.S. Swelling-activated organic osmolyte efflux: a new role for anion channels. Kidney Int. 48:994– 1003;1995. 36. Surratt, C.K.; Wang, J.-B.; Yuhasz, S.; Amzel, M.; Kwon, H.M.; Handler, J.S.; Uhl, G.R. Sodium- and chloride-dependent transporters in brain, kidney, and gut: lessons from complementary DNA cloning and structure-function studies. Curr. Opin. Nephrol. Hypertens. 2:744–760;1993. 37. Takenaka, M.; Bagnasco, S.M.; Preston, A.S.; Uchida, S.; Yamauchi, A.; Kwon, H.M.; Handler, J.S. The canine betaineGABA (gamma-amino-n-butyric acid) transporter gene. Diverse mRNA isoforms are regulated by hypertonicity and are expressed in a tissue specific manner. Proc. Natl. Acad. Sci. USA 92:1072 –1076;1995. 38. Takenaka, M.; Preston, A.S.; Kwon, H.M.; Handler, J. 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. 39. Terada, Y.; Tomita, K.; Homma, M.K.; Nonoguchi, H.; Yang, T.; Tamada, T.; Yuasa, Y.; Krebs, E.G.; Sasaki, S.; Marumo, F. Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase by
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