Biochemistry, Hypertonicity

Fundamentals

Disturbances in tonicity are the major clinical disorders affecting volume, proper function, and survival of cells. It causes water movement into or out of cells, thereby diluting or concentrating intracellular ions.[3] Numerous mechanisms use the movement of water for cells to maintain their homeostatic size and functioning. Hyperosmolality itself alters several intracellular processes, including cell volume regulation, cell cycle, intracellular ion homeostasis, macromolecular and nucleic acid stability, and can induce apoptosis.[4]

Issues of Concern

When typical mechanisms of homeostasis are unable to regulate tonicity, cell damage can occur secondary to prolonged hypertonicity and from fast onset hypertonicity. Clinically, hyperglycemic and hypernatremic states are the main etiologies for disease-causing hypertonicity. Cell shrinking secondary to hypertonicity can cause severe clinical manifestations and even death.[1] 

Cellular Level

In a hypertonic environment, cells use membrane proteins called aquaporin channels to take advantage of the osmotic pressure gradient and shift water towards the higher concentration medium. Cells are permeable to water, and thanks to this, they can shrink and elevate the concentration of intracellular solutes. Cell survival mechanisms include the accumulation of organic osmolytes and increased expression of proteins through numerous pathways, resulting in osmotolerance.[5] The shrinkage of cells generates stress that is commonly adjusted pathways involving Na+, K+-ATPase (in steady conditions). "Fast" volume regulation due to rapid activation of membrane ion transporters and "slow" adaptation to chronic changes in extracellular osmolarity involving modifications in gene expression and intracellular organic osmolyte content.[6]

In steady-state conditions, a fundamental property of cells is that they contain a significant amount of large-molecular-weight anionic colloids, mostly proteins and organic phosphates, to which the plasma membrane is impermeable.[7] The restriction of these proteins within a compartment that is impermeable to them allows for the Donnan effect. The Donnan effect describes the production of a higher concentration due to the inability of certain particles to cross a semi-permeable membrane, generating osmotic forces between the extracellular and intracellular compartment.[8]

Molecular Level

Hypertonicity denotes a relative excess of the solute with extracellular distribution over body water regardless of whether body water is normal, reduced, or excessive. The gain of extracellular solutes leads to the osmotic exit of water from the intracellular compartment to dilute the extracellular solutes. Sodium salts, including sodium chloride and sodium bicarbonate, are the major extracellular solutes and routinely indicate hypertonicity when elevated.[9]

Under hypertonic conditions, ions such as Na, Cl, and K accumulate in the cytosol and get exchanged for compatible organic osmolytes that do not perturb intracellular protein structure or function.[10] A fast response is driven by the rapid activation of Na+, K+, 2Cl- co-transporter, and the Na+/H+ exchanger, which couples to Cl-/H2CO3- anion exchanger.

On the other hand, TonEBP (tonicity-responsive enhancer-binding protein), also known as NFAT5 or OREBP, is a transcription factor that can promote the cellular accumulation of organic osmolytes in the hypertonic renal medulla. This is done by stimulating the expression of its target genes in the kidneys, but it is also abundantly expressed in the brain, heart, liver, and activated T-cells.[11][12]

Additionally, there is evidence that osmotic stress elicits a morphological disruption of the transverse tubular system in skeletal muscle fibers. The transverse tubular system is a continuation of the surface membrane that forms a junction with the sarcoplasmic reticulum and is known for its calcium storage. It is the primary interface between the myoplasm and the extracellular environment, and these arrangements are essential for muscle contraction.[13]

Function

Regulation of osmolarity and volume play an essential role in maintaining body water balance and tonicity.[2]

The acute adaptation to hypertonicity consists of ''regulatory volume increase'' (RVI). It requires activating the Na+, K+, 2Cl- co-transporter, and the Na+/H+ exchanger, which couples to the Cl-/H2CO3- anion exchanger. These last two bring NaCl and KCl into the cell and moves H2CO3 out of it. The H2CO3 is then converted to CO2 and returns to the pool of H+ and HCO3- inside the cells. The thermodynamically obliged movement of water also follows the return of H2CO3. Sodium-ion entering the cells is extruded through Na/K ATPase in exchange for potassium, forming potassium chloride, the final salt gained intracellularly in hypertonicity.[1]

Meanwhile, with chronic adaptation, the general response to hypertonicity is activating the transcription factor tonicity (TonEBP), leading to increased cellular expression of organic osmolyte transporters and enzymes. Some of the transcriptions of genes that TonEBP produces are aldose reductase (AR), betaine/GABA transporter(BGT1), sodium myoinositol transporter (SMIT), and taurine transporter (TauT). The transcription of Hsp70, urea transporters (UTA1 and -2), and water channel aquaporin-2 (AQP2) increase cell membrane water permeability and is activated by TonEBP.[14]

Mechanism

Cells that are shrunken by hypertonicity respond initially with RVI. It increases the uptake of inorganic salts and the osmotic influx of water but results in high intracellular inorganic salt concentration that can perturb cellular function and structure. To counteract this, cells activate TonEBP for the transcription of genes for aldose reductase (for sorbitol synthesis) and transporters of betaine, inositol, and taurine. This process accumulates large amounts of organic osmolytes, which provide an osmoprotective effect.

Most cells in mammals are generally not stressed by hypertonicity because of the close control of NaCl concentration in virtually all extracellular body fluids. The renal inner medulla is a striking exception.[15] This is because of its urinary concentrating mechanism, where it has routine exposure to extremely high concentrations of sodium chloride (NaCl) and urea. The adaptation of medullary cells to hyperosmotic stress involves acute cellular efflux of water, cell shrinkage by NaCl, chronic accumulation of compatible organic osmolytes, and acute activation of immediate-early and heat shock genes.[16] Renal medullary cells do not restrict this mechanism, which also happens in other tissues when exposed to pathologic conditions that produce hypertonic states.

Testing

The predominant clinical syndromes of hypertonicity are hypernatremia and hyperglycemia.[9] Rises in tonicity from changes in body water, body solute, or both can be assessed by testing osmolarity in serum and urine and correlating it with the level of electrolytes in these two compartments to establish the cause of the impairment. The serum osmolarity normal range is 280 to 295mOsm/ kgH2O and normal urine osmolality is from 50mOsm/kgH2O to 1400mOsm/kgH2O.[2] Normal serum sodium is 135 to 145mmol/L, and the urinary sodium reference range varies with the diet.[17]

Pathophysiology

Tonicity is tightly regulated by the equilibrium between water intake and water excretion.[2] Normal conditions where water loss occurs are respiration, within gastrointestinal fluids, in urine, and through the skin. The problem occurs when patients are unable to replete those losses. When the osmoreceptors in the hypothalamus sense the increase in serum tonicity, water intake is suggested by the stimulation of thirst. In addition, the kidney's primary reaction to water loss is through concentrating the urine. Just 1% of the change in tonicity is enough to produce ADH release, but it needs a greater than 10% fall in extracellular volume to be released. ADH acts on the V2 receptors in the principal cells of the collecting tubules within kidneys and causes the expression of aquaporins for water movement from the tubules to the hypertonic interstitium.

One of the cardinal manifestations of a hyperglycemic crisis is hypertonicity.[18] The excess of glucose in the extracellular fluid has a hypertonic effect and produces an osmotic diuresis that can cause water loss to exceed the losses of sodium and potassium. This results in an elevated sodium concentration within the cell and will stimulate thirst.

High-glucose conditions in patients with diabetic microvascular complications, particularly with diabetic nephropathy, have shown TonEBP to upregulate the expression of AR.[19] The production of AR in cells that can produce the enzyme is desired for the enzyme's ability to catalyze the reaction of glucose to sorbitol. The inability of sorbitol to cross cell membranes and its accumulation within the cell aids in counteracting the osmotic stress placed on cells during a hyperglycemic event. AR is present in tissues such as nerves, retina, lens, glomerulus, and vascular cells.[20]

During acute states of tonicity variability, the brain is also in danger. The primary defensive adaptation occurs through RVI, but astrocytes also play a major role by accepting the movement of water from the cerebrospinal fluid.

Clinical Significance

Acute hypertonicity most often affects children and the elderly more than other ages. Patients commonly develop fever, nausea, and vomiting. In children, symptoms can range from irritability, restlessness, muscular twitching, hyperreflexia, and seizures. In the elderly, seizures are rarely present, but patients can present with lethargy, delirium, and become comatose. On the other hand, chronic hypertonicity may manifest with subtle neurological changes because it has more time to adapt to the medium, even when severe hypertonicity is severe.[9]

Conditions causing hypernatremia due to inadequate water intake are associated with:[9]

• Lack of water sources
• A central nervous system disorder compromising neural pathways of thirst
• Tracheal intubation
• Sedation
• Dementia
• Delirium
• Paranoia
• Severe depression
• Degenerative processes like Parkinson's disease
• Diabetes insipidus

It can also result from excessive water loss, such as in:[9]

• Gastrointestinal losses
• Excessive sweating
• Mechanical ventilation
• Glucosuria
• Diabetes insipidus
• Genetic mutation of V2 receptor gene or aquaporin 2 gene
• Medication-induced diuresis

Glucose is an osmotically active substance that causes the movement of water out of the cells and subsequently reduces serum sodium levels by dilution. Therefore it is crucial to correct serum sodium for hyperglycemia, calculated by adding to measured [Na] 1.6 mmol/L for every 100 mg/dL (5.55 mmol/L) increment of serum glucose above normal.[21] Under other conditions, uncontrolled hyperglycemic patients produce osmotic diuresis, losing water and then causing a hypovolemic state presenting with signs such as orthostatic hypotension, increased pulse rate, decreased skin turgor, flat neck veins, and dry mucous membranes. The osmotic diuresis during uncontrolled hyperglycemia can ultimately lead to hypernatremia if there is an insufficient replacement for this water loss. For that reason, in patients with diabetes mellitus, sodium concentration can be variable on presentation. This is due to the hyperglycemia-induced water movement out of the cells that lower Na and the glucosuria-induced osmotic diuresis.