hepoxilin A. 3 and the 5-lipoxygenase product leukotri- ate carboxykinase (PEPCK), tyrosine aminotransfer- ase, aB crystallin, laminin B. 2 and matrix proteins.
Nephrol Dial Transplant (1998) 13: 867–874
Nephrology Dialysis Transplantation
Molecular Basis of Renal Disease
Mechanisms and clinical significance of cell volume regulation Siegfried Waldegger1, Silvia Steuer2, Teut Risler2, August Heidland3, Giovambattista Capasso4, Shaul Massry5 and Florian Lang1 Departments of 1Physiology and 2Internal Medicine, University of Tu¨bingen, 3Kuratorium fu¨r Nephrologie, Wu¨rzburg, Germany, 4Department of Nephrology, Second University of Napoli, Italy and 5Division of Nephrology, University of Southern California, Los Angeles, CA
Abstract A wide variety of factors challenge constancy of cell volume. Alterations of cell volume activate diverse cell volume regulatory mechanisms including ion transport, osmolyte accumulation, metabolism and expression of appropriate genes. A wealth of cellular signalling pathways link cell volume to the respective regulatory mechanisms. Cell volume emerges as a pathophysiologically important parameter in several diseases including diabetes mellitus, uraemia, hepatic insufficiency and hypercatabolic states. The role of altered cell volume in disease is a challenge which requires more experimental research and clinical investigation.
Introduction One of the most important tasks of the kidney is to finely tune water and electrolyte composition of the body. Without appropriate water and electrolyte homeostasis constancy of body compartments cannot be achieved. Thus, failure of the kidney adequately to regulate water and electrolyte excretion must eventually lead to alterations of extracellular and/or intracellular volumes. While a great deal of information has been gathered on causes and sequelae of deranged extracellular volume homeostasis, the pathophysiology of cellular volume homeostasis has remained less understood. Even at constant extracellular osmolarity a wide variety of mechanisms challenge cell volume homeostasis and a similar diversity of mechanisms serve to maintain a constant cell volume. Beyond that alterations of cell volume profoundly influence a number of cellular functions not obviously related to cell volume. We provide a brief discussion of the challenges of cell volume constancy, cell volume regulatory mechanisms and cell volume-sensitive functions as well as Correspondence and offprint requests to: Prof. Dr F. Lang, Physiologisches Institut, Universita¨t Tu¨bingen, Gmelinstrasse 5, D-72076 Tu¨bingen, Germany.
some examples illustrating the participation of deranged cell volume regulation in disease states. For a more extensive treatment of cell volume regulatory mechanisms and the functional significance of cell volume the reader is referred to previous reviews [1–3].
Challenges of cell volume constancy With only few exceptions, cell membranes are highly permeable to water [4]. In general, water movement is driven by an osmotic and a hydrostatic pressure gradient. However, animal cell membranes do not withstand significant hydrostatic pressure gradients and water movement is determined by osmotic gradients across the cell membrane. To avoid alterations of cell volume, cells have to maintain osmotic equilibrium across the cell membrane. Excess intracellular osmolarity will lead to entry of water and thus cell swelling, whereas excess extracellular osmolarity will abstract water from the cells and thus lead to cell shrinkage. Since cells accumulate osmotically active substrates, such as amino acids and carbohydrates, they must decrease intracellular electrolyte concentration to achieve osmotic equilibrium across the cell membrane [5]. To this end cells extrude Na+ in exchange for K+ by the Na+/K+ ATPase. Usually the cell membrane is poorly permeable to Na+ but highly permeable to K+. According to the chemical gradient K+ tends to exit the cell through K+ channels creating a cell-negative potential difference across the cell membrane. This cell membrane potential drives exit of anions such as Cl−. As a result, Cl− is some 80 mM lower within as compared to outside of the cell, thus establishing osmotic equilibrium. As long as the cell membrane is not perfectly impermeable to Na+, the maintenance of a constant cell volume requires expenditure of energy to fuel the operation of Na+/K+ ATPase. As described later in this review, energy depletion will eventually lead to cell swelling. Constancy of cell volume is further challenged by ion channel activation. Activation of Na+ channels depolarizes the cell membrane and thus favours simul-
© 1998 European Renal Association–European Dialysis and Transplant Association
868
taneous entry of Cl−. The cellular accumulation of NaCl leads to cell swelling. On the other hand, activation of K+ and/or Cl− channels will favour KCl exit and results in cell shrinkage. The channels are activated by a wide variety of mediators. Glutamate, for instance, swells some cells by activation of Na+ channels [6 ] and acetylcholine shrinks cells by activation of K+ and Cl− channels [7]. Increase of extracellular K+ concentration decreases the chemical driving force for K+ exit, depolarizes the cell membrane and thus favours cellular KCl accumulation and cell swelling [2]. Activation of Na+, K+, 2Cl− co-transport leads to cellular uptake of NaCl and KCl and thus to cell swelling. Stimulation of Na+/H+ exchange leads to entry of Na+ and intracellular alkalosis which in turn stimulates Cl−/HCO− exchange. The tandem leads 3 to NaCl accumulation, since H+ and HCO− are 3 replenished within the cells from CO (or H CO , 2 2 3 respectively) which readily permeates across the cell membrane [3]. Na+, K+, 2Cl− co-transport and Na+/H+ exchange are activated by insulin [8–10] and a wide variety of growth factors [11], which thus lead to cell swelling. Glucagon and cAMP shrink hepatocytes by stimulation of ion channels [8,9]. Organic acids, such as lactate may enter the cell in the non-dissociated moiety, dissociate within the cell and thus create intracellular acidosis. H+ ions are then extruded by the Na+/H+ exchanger and the cells swell due to accumulation of Na+ and the acid anion [2]. Na+-coupled concentrative uptake of substrates such as amino acids or glucose leads to depolarization, parallel entry of Cl− and cell swelling due to accumulation of NaCl and the substrates [12,13]. On the one hand breakdown of proteins, glycogen or triglycerides, which are osmotically less active than the sum of their constituent parts leads to an increase, on the other hand release of substrates and formation of proteins, glycogen or triglycerides to a decrease of intracellular osmolarity. Obviously, alterations of extracellular osmolarity challenge cell volume constancy and clinical conditions with hypo- or hypernatraemia may be associated with cell swelling and cell shrinkage respectively, as outlined below.
S. Waldegger et al.
(a)
Cell volume regulatory mechanisms
(b)
Alterations of cell volume trigger a variety of cellular mechanisms aiming at re-establishing the set point of cell volume. Among these, ion transport across the cell membrane is the most rapid and efficient mechanism of adjusting cellular osmolarity.
Fig. 1. Cell volume regulatory mechanisms. (A) Mechanisms increasing cell volume following cell shrinkage: Parallel activation of Na+/H+ exchanger and Cl−/HCO− exchanger (a); Na+, K+, 3 2Cl− cotransport (b); NaCl cotransport (c); Na+ channels and Na+/K+ ATPase (d); inhibition of K+- and Cl− channels (e); cellular accumulation of glycerophosphorylcholine by inhibition of phosphodiesterase (f ), Na+ coupled accumulation of inositol, taurine and betaine (g); cellular accumulation of sorbitol by activation of aldosereductase (h). (B) Mechanisms decreasing cell volume following cell swelling: Activation of K+ and anion channels (a); activation of KCl cotransport (b); activation of Na+/Ca++ exchanger and Ca++ ATPase (c); parallel activation of K+/H+ exchange and Cl−/HCO− exchange (d); release of osmolytes, such 3 as sorbitol, inositol, taurine, betaine (e); Na+ ATPase (f ); inhibition of Na+ channels (g).
Cell volume regulatory ion transport Following cell swelling, cells have to release ions to reduce their osmolarity. In most cells ion release is accomplished by activation of K+ channels and/or anion channels ( Figure 1). A great diversity of distinct
Cell volume regulation
ion channels has been shown to serve volume regulation in different cells. Other transport processes mediating cellular loss of electrolytes include KCl symport, parallel K+/H+ exchange and Cl−/HCO− exchange 3 ( leading to KCl loss) as well as Na+/Ca2+ exchange in parallel to Ca2+ ATPase ( leading to loss of Na+) [3]. Following cell shrinkage, cells accumulate ions through activated Na+, K+, 2Cl− cotransport and/or Na+/H+ exchange together with Cl−/HCO− exchange 3 (see above). The Na+ thus accumulated is replaced by K+ via the Na+/K+ ATPase. The transporters thus mediate eventually uptake of KCl. Shrunken cells avoid further ion loss by inhibition of K+ and/or Cl− channels [3]. Osmolytes The use of ions as osmolytes is limited, since high ion concentrations interfere with the stability of proteins. Moreover, alterations of intracellular Na+ activity could increase intracellular Ca2+ activity via reversal of Na+/Ca2+ exchange, and the stimulation of Na+/H+ exchange in shrunken cells usually results in cellular alkalinization. Altered intracellular pH and Ca2+ concentration in turn affect a multitude of cellular functions. To avoid the effects of altered ion concentrations, most cells utilize so called compatible osmolytes, i.e. organic substances specifically designed to create intracellular osmolarity without destabilizing proteins [14–17]. The most important osmolytes are polyols such as sorbitol and myoinositol, the amino acid taurine and methylamines, such as betaine and glycerophosphorylcholine. Glycerophosphorylcholine (GPC ) formation from phosphatidylcholine is catalysed by a phospholipase A other than the arachidonyl selective enzyme, and is 2 degraded by a phosphodiesterase to glycerol-phosphate and choline. Increase of extracellular osmolarity inhibits the phosphodiesterase and thus leads to accumulation of GPC. Sorbitol is produced from glucose by an aldose reductase. Osmotic cell shrinkage stimulates the transcription rate of the aldose reductase. Myoinositol (inositol ), betaine and taurine are accumulated by specific Na+-coupled transporters. Osmotic cell shrinkage stimulates the transcription rate of the transport molecules. Similarly, some amino acids are accumulated into shrunken cells by stimulation of Na+-coupled transport. Upon cell swelling GPC, sorbitol, inositol, betaine and taurine are rapidly released through poorly defined transport systems (for review see Lang et al. [2,3]).
869
proteolysis and glycogenolysis [13,18–26 ]. The amino acids and glucosephosphate are thus incorporated into the osmotically less active macromolecules. The effect of cell volume on protein metabolism is exploited by hormones such as insulin and glucagon ( Figure 2). As a matter of fact, the antiproteolytic effect of insulin and the proteolytic effect of glucagon in the liver is exclusively the result of the respective swelling or shrinking effect of the hormones [8–10]. A number of further metabolic pathways have been shown to be sensitive to cell volume. Cell swelling inhibits glycolysis and stimulates flux through the pentose phosphate pathway, NADPH production [27,28], glutathione (GSH ) formation and efflux into blood [29], glycine oxidation [30], glutamine breakdown [12], formation of NH+ and urea from amino 4 acids [31], ketoisocaproate oxidation [30] as well as lipogenesis from glucose [32]. The mRNA for phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, is decreased by cell swelling [33]. Signalling Alterations of cell volume influence a myriad of cellular signalling molecules which in turn trigger the cell volume regulatory machinery. Cell swelling may lead to increase of intracellular Ca2+ activity by cellular release triggered by 1,4,5inositoltrisphosphate and/or Ca2+ entry through Ca2+ channels in the plasma membrane [34]. Cell volume changes modify the architecture of the cytoskeleton and the expression of cytoskeletal proteins [35,36 ]. Conversely, cytoskeletal elements participate
Other metabolic pathways sensitive to cell volume Cell shrinkage stimulates the breakdown of proteins and of glycogen to amino acids and glucosephosphate, respectively, which are osmotically more active than the macromolecules. Cell shrinkage inhibits protein and glycogen synthesis. Conversely, cell swelling stimulates protein and glycogen synthesis and inhibits
Fig. 2. Effect of insulin and of glucagon on cell volume. Insulin swells cells by KCl uptake via activation of Na+/H+ exchange, Na+, K+, 2Cl−-cotransport and Na+/K+ ATPase. Glucagon shrinks cells by activation of K+- and anion channels. The cell volume changes participate in the signalling of hormone action. For instance, the antiproteolytic effect of insulin and the proteolytic effect of glucagon completely depend on the hormone induced alterations of cell volume.
870
S. Waldegger et al.
Fig. 3. Cell volume regulated gene expression. Cell volume influences the expression of a variety of genes. It does so in part through osmoresponsive or cell volume responsive elements (CVRE ). Genes sensitive to cell volume encode for carriers, cytoskeletal elements, enzymes and signaling molecules. The insert demonstrates the upregulation of the serine/threonine kinase h-sgk by increase of medium osmolarity.
in the triggering of cell volume regulatory mechanisms [33,37–39]. Both cell swelling and cell shrinkage modify the phosphorylation of a variety of proteins, kinases reported to be activated by cell swelling include tyrosine kinases, protein kinase C, adenylate cyclase, MAP kinase, Jun-kinase, and focal adhesion kinase (p121FAK ) [1]. Osmotic cell shrinkage has been postulated to activate protein kinase C or a similar kinase. As a matter of fact, cell shrinkage increases the expression of the serine/threonine kinase h-sgk (human serum and glucocorticoid-dependent kinase) [40]. The targets of this kinase, however, have not been identified yet. Cell swelling activates a phospholipase A , leading 2 to formation of the 15-lipoxygenase product hepoxilin A and the 5-lipoxygenase product leukotri3 ene LTD [1]. These eicosanoids are thought to trigger 4 cell volume regulatory K+ and/or Cl− channels and/or volume regulatory taurine release. The enhanced formation of leukotrienes may parallel a decreased formation of PGE leading to inhibition of PGE 2 2 sensitive Na+ channels. On the other hand, PGE may 2 activate volume regulatory K+ channels in other cells [2]. Cell swelling alkalinizes acidic cellular compartments, whereas cell shrinkage enhances the acidity in these compartments [37,41,42]. In hepatocytes, this effect may contribute to the antiproteolytic action of cell swelling since lysosomal proteases are known to have acidic pH optima and lysosomal alkalinization is known to inhibit proteolysis [43]. Experiments in other
cells such as pancreatic b cells and neurons demonstrate that cell swelling alkalinizes similarly secretory granules [44,45]. Cell volume influences the expression of several genes (Figure 3). As indicated above, cell shrinkage stimulates expression of enzymes or transporters accomplishing cellular formation or accumulation of osmolytes, such as the aldose reductase, and the Na+coupled transporters for betaine, taurine, inositol and amino acids as well as Na+, K+, 2Cl− co-transport. The enhanced expression of the cell volume regulated kinase h-sgk possibly triggers mechanisms of regulatory cell volume increase. Other genes preferably expressed in shrunken cells include ClC-K1, P-glycoprotein, heat shock proteins, the immediate early genes Egr-1 and c-fos, cycloxygenase-2, phosphoenolpyruvate carboxykinase (PEPCK ), tyrosine aminotransferase, aB crystallin, laminin B and matrix proteins. 2 Genes expressed preferably in swollen cells include bactin, tubulin, immediate early gene c-jun, ornithine decarboxylase, arginine succinate lyase and tissue plasminogen activator [2,46 ].
Pathophysiology of cell volume Energy depletion Constancy of cell volume is compromised by energy depletion, which will dissipate the Na+ and K+ gradients, lead to depolarization and cellular accumulation of Cl− ( Figure 4). Furthermore, an increasing extracel-
Cell volume regulation
871
Fig. 4. Cell swelling by energy depletion. The Na+/K+ ATPase maintains a low intracellular Na+ and high intracellular K+. K+ diffusion along the chemical gradient establishes a cell membrane potential (PD) which drives Cl− out of the cells (A). Energy depletion compromizes the function of the Na+/K+ ATPase, and thus leads to dissipation of Na+ and K+ gradient, depolarization, entry of Cl− and subsequent cell swelling (B).
lular K+ concentration will depolarize the cell membrane leading to Cl− accumulation [47]. The cell swelling during anoxia is compounded by the formation of lactate, cellular acidosis and enhanced Na+/H+ exchange activity (see above). In neuronal tissue, the depolarization triggers the release of glutamate and the subsequent opening of unspecific cation channels results in further cell swelling [2,47]. Cell proliferation and apoptotic cell death Growth promoters are well known stimulators of Na+/H+ exchange and some growth factors have been described to stimulate Na+, K+, 2Cl− co-transport [11,48]. Similarly, ras oncogene, which allows growth factor independent cell proliferation has been shown to shift the set point for cell volume regulation towards greater cell volume [11,49]. Activation of Na+/H+ exchange leads in addition to cellular alkalinization [50–53] and cell swelling to an increase of pH in lysosomes of proliferating cells [54]. Cell volume increase and subsequent alkalinization of lysosomal pH may account for the antiproteolytic action of growth factors, such as TGF-b1. One of the hallmarks of apoptotic cell death is cell shrinkage. Indeed, marked osmotic cell shrinkage (>30%) has been shown to trigger apoptotic cell death. However, a moderate decrease of cell volume (
